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- Pubmed Central
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- Copyright © 2017, The Authors
- ISSN
- 2375-2548
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- 2375-2548
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- 10.1126/sciadv.1601966
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Abstract
SCIENCE ADVANCES RESEARCH ARTICLE NANOELECTRONICS 2017 © The Authors, some rights reserved; exclusive licensee Ultraflexible nanoelectronic probes form reliable, glial American Association for the Advancement scar–free neural integration of Science. Distributed under a Creative 1 2 2 3 2 Lan Luan, * Xiaoling Wei, * Zhengtuo Zhao, * Jennifer J. Siegel, Ojas Potnis, Commons Attribution 2 2 2 2 2 Catherine A Tuppen, Shengqing Lin, Shams Kazmi, Robert A. Fowler, Stewart Holloway, NonCommercial 2 3 2† Andrew K. Dunn, Raymond A. Chitwood, Chong Xie License 4.0 (CC BY-NC). Implanted brain electrodes construct the only means to electrically interface with individual neurons in vivo, but their recording efficacy and biocompatibility pose limitations on scientific and clinical applications. We showed that nanoelectronic thread (NET) electrodes with subcellular dimensions, ultraflexibility, and cellular surgical footprints form reliable, glial scar–free neural integration. We demonstrated that NET electrodes reliably de- tected and tracked individual units for months; their impedance, noise level, single-unit recording yield, and the signal amplitude remained stable during long-term implantation. In vivo two-photon imaging and postmor- tem histological analysis revealed seamless, subcellular integration of NET probes with the local cellular and vasculature networks, featuring fully recovered capillaries with an intact blood-brain barrier and complete ab- sence of chronic neuronal degradation and glial scar. INTRODUCTION Taking previous efforts in improving the neural electrode interface Chronically implanted electrodes (1–4) enable one of the most impor- into consideration, we identified the following key aspects for estab- tant neurotechniques by allowing for the acquisition of individual neu- lishing a reliable and glial scar–free neural-probe interface: (i) the ronelectricalactivitiesinthe living brain(5–7). Although long-term dimension of the probe is comparable to or smaller than that of av- electrical recording was sometimes observed (2, 8), large variations in erage cells and capillaries, such that its perturbation to the host chronic recording capacity were often reported (3, 4), largely due to biological matrix is minimal (10, 17); (ii) the probe has sufficient flex- the lack of stability at the interface between conventional electrodes ibility to ensure complete compliance to tissue micromovements and and the brain tissue (4, 9) in both the short and the long term. to reduce probe-tissue interfacial force to the range of cellular force Conventional neural implants have volumes and surgical footprints (nanonewtons) (20, 23); (iii) the surgical damage during implantation considerably larger than those of cells and capillaries, which induce is less than ca. 100 mm across to allow for tissue recovery (24, 25); and substantial damage and disruption to local cellular and vascular net- (iv) the probe is mechanically and electrically robust for long-term works (10). Moreover, these probes are significantly more rigid than functionality under physiological conditions. To meet these stringent the host brain tissue, whose natural micromovements induce intense requirements, we developed ultraflexible nanoelectronic thread (NET) stress at the interface (11). In the short term, the resulting electrode brain probes and an implantation strategy with cellular-sized surgical displacement from their targeted neurons leads to sudden waveform footprints. We verified the effectiveness of our approach in a rodent changes in time scales as short as hours that prevent reliable tracking (mouse) model by chronic electrical recording and comprehensive of individual neuronsoverdaysand longer (12, 13). In the long term, characterizations of the probe-tissue interface. the presence of implants causes recurring cellular and vascular dam- age, elicits sustained inflammation and tissue response (14), and even- tually leads to neuronal degradation and glial scar formation near the RESULTS implants (10, 15, 16). These chronic deteriorations are manifested in We designed and fabricated two types of NET brain probes, NET-50 electrical recordings as degradations in recording fidelity, including and NET-10, using a multilayer, substrate-less architecture and increasing impedance, elevated noise levels, decreasing signal ampli- specialized photolithography (Materials and Methods). As shown in tudes, and diminishing unit recordings (1–4, 16). Fig. 1, NET-50 had a four-layer layout with a total thickness of Extensiveresearchefforts in thepastdecadehavedemonstratedthat 1 mm and an average width of 50 mm, hosting a linear array of eight reducing the neural probe dimension (17) and rigidity (16, 18, 19) electrodes. NET-10 featured a cross section of 10 mm×1.5 mm, the improves neural interface. Recent work has shown that macroporous smallest among all reported neural probes to the best of our knowl- electronics (20) and ultrasmall microelectrodes made of carbon fibers edge, hosting four electrodes on two opposite surfaces in a seven-layer (21, 22) greatly reduced tissue response. However, reliable brain probes layout. To minimize volume and maximize flexibility, both types had that detect and track activities from the same neurons for extended electrodes and interconnects on different layers electrically connected periods with none of the aforementioned chronically detrimental effects by vias through the insulation layers. NET-50 probes were designed require seamless biointegration but are yet to be demonstrated. analogous to the commonly used silicon neural probe (for example, a single slab in NeuroNexus A8×8 design), whereas NET-10 probes were designed to offer similar recording characteristics to tetrodes (1, 3), both with aggressively reduced dimensions (fig.S1).Compared Department of Physics, the University of Texas at Austin, TX 78712–1192, USA. Department of Biomedical Engineering, the University of Texas at Austin, TX with previously demonstrated neural probes (18, 21, 26), the NET 78712–1192, USA. Center for Learning and Memory, Institute for Neuroscience, probes drastically reduced the effective bending stiffness and tissue the University of Texas at Austin, TX 78712–1192, USA. displacement per electrode for markedly improved biocompatibility *These authors contributed equally to this work. †Corresponding author. Email: [email protected] (fig.S2).Specifically,the bending stiffness was reduced by orders of Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 1of9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 1. Structures of NET neural probes. (A and B) As-fabricated NET-50 and NET-10 probes on substrates. (C and D) Zoom-in views of two electrodes as marked by the dashed boxes in (A) and (B), respectively. Arrows denote “vias.” (E) Schematics of the probe cross section in (A, top) and (B, bottom), highlighting the multilayer layout. Color code: gray, insulation; orange, interconnects; and blue, electrodes. Not drawn to scale. (F) Height profile of the NET-50 probe along the dashed line in (C) measured by an atomic force microscope. (G) A NET-50 probe suspended in water. A knot is made with a curvature of less than 50 mm to illustrate its flexibility and robustness. (H) Multiple NET-10 probes suspended in water. Arrows denote the probes. Scale bars, 100 mm(A),50 mm (B, G, and H), and 10 mm(Cand D). −15 2 magnitude to 10 N·m , which brought down the probe-tissue inter- dimensions by using a temporary engaging mechanism (Fig. 2A) and facial force to the nanonewton range (fig. S3), on par with the single- shuttle devices made of carbon fibers and tungsten microwires with diam- cell traction forces (27). eters as small as 7 mm(Fig. 2B). Theengaging mechanism was enabled The ultraflexibility of the NET probes mechanically precluded their by a micropost at the end of each shuttle device fabricated using focused self-supported penetration through brain tissue. Previous strategies for ion beam (FIB) (fig. S4). During delivery, the micropost engaged into delivering flexible probes included temporarily altering the probe’sri- the microhole (Fig. 2, C to E) and pulled the NET probe to the desired gidity (20) and, more commonly, temporarily attaching the probe to a depth (movie S1), after which the shuttle device disengaged and rigidshuttledevice(18, 19, 23, 28–31). However, most shuttle devices retracted (Materials and Methods). The overall insertion footprint used in previous studies had dimensions larger than 100 mm, which was as small as ca. 10 mm across (Fig. 2, D and E), which led to only were significantly larger than the dimensions of the NET probes and cellular-sized surgical damage, as evidenced by little bleeding (Fig. 2, resulted in unrecoverable damage and persistent scarring (24, 25). We Fand G) andsmall insertionforce (fig.S4).Takingadvantage of reduced the implantation footprints by about 10-fold to the cellular the ultraflexibility and the ultrasmall dimensions, we accommodated Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 2of9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 2. Implantation procedure for the NET probes. (A) Schematic showing the temporary engaging mechanism. Arrows denote the entry site of the implantation (solid), the delivery path of the shuttle device (gray), and the path of the engaged NET probe (dashed). Inset: Zoom-in view of the dashed square highlighting that the micropost engages in the microhole on the NET probe at the end of the shuttle device. (B) Photograph of a typical carbon fiber shuttle device, with a diameter of 7 mm and a length of 3 mm, mounted at the end of a micromanipulator. Scale bar, 500 mm. Inset: Scanning electron microscopy (SEM) image of the micromilled post with a diameter of 2 mmand aheight of 5 mm at the shuttle device tip. Scale bar, 2 mm. (C) Optical micrographs showing engaging holes in NET-50 (top) and NET-10 (bottom) probes with a slightly larger diameter than the post, as denoted by the arrows. (D and E) False-colored SEM images of NET-50 and NET-10 probes (green) attached on shuttle devices with a 20-mmtungsten microwire (D, purple) and a 10-mm carbon fiber (E, purple) showing their ultrasmall dimensions. Scale bars, 50 mm (D) and 20 mm(E). (F and G) Micrographs showing that both NET-50 and NET-10 probes were successfully delivered into the living mouse brain with minimal acute tissue damage. Arrows denote the delivery entry sites. Scale bars, 100 mm (F) and 50 mm(G). (H) Schematic of skull fixation that accommodates both connectors for the neural probes and a glass window allowing optical access. Not drawn to scale. (I) Photograph of a typical postsurgery mouse with implanted NET probes and a glass window mounted on top. Insets: top, image of a cable connector mounted on the skull; bottom, zoom-in view of the glass window in which the arrow denotes an implanted probe. (J) Typical unit activities recorded by eight electrodes on an implanted NET-50 probe. A high-pass filter (300 Hz) was applied. chronic optical access with NET implants (Fig. 2, H and I), which ber of electrodes that detected unit events and sortable single-unit allowed for monitoring of the tissue-probe interface by in vivo action potentials (APs) increased in the first 1.5 months and then two-photon (2P) imaging. The electrical integrity was maintained remained at the same level (Fig. 3B; examples of unit activities that throughout surgery and the implanted electrodes readily detected include both nonsortable spikes and sortable single-unit APs are unit activities (Fig. 2J). detailed in fig. S5). The yields of multi-unit (~75%) and sortable We next evaluated the long-term reliability of the NET probes. We single-unit recording (~25%) were on par with conventional silicon implanted 16 probes into the somatosensory and visual cortices of seven probes for spontaneous measurement under anesthesia (32), but with mice, containing a total of 80 of the 96 connected electrodes (83.3% fab- unprecedented chronic stability. Third, we detected sortable AP wave- rication yield). We performed electrical recording on anesthetized forms from 19 electrodes with average amplitude and signal-to-noise animals twice a month for 4 months (Materials and Methods). We ob- ratio (SNR) being stable throughout the 4-month period (Fig. 3C). served that recording performance improved during the initial 1.5 months We followed all 80 electrodes for 4 months and summarized their and then remained stable for at least another 2.5 months (until the animal- and probe-specific performanceovertimeintable S1 andfig. conclusion of our experiment period) in the following aspects: First, S5. Although there were variations in the number of functional electro- the average impedance and the noise level of all 80 electrodes decreased des and the unit detection yield among different animals, stability and in the first 1.5 months and remained stable (Fig. 3A). Second, the num- consistency in unit detection over time were observed in all animals. As Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 3of9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 3. Chronic recording and electrical characterization of implanted NET electrodes. (A) Impedance (red) and noise level (blue) of 80 implanted electrodes as a function of time. Error bars mark the SD. (B) The number (left) and percentage (right) of electrodes that recorded unit activities (red) and sortable single-unit APs (orange) as a function of time. (C) Average peak-valley amplitude (red) and SNR (blue) of single-unit APs recorded by n = 19 electrodes as a function of time. Error bars indicate the SD. (D) Twice-a-month measurements for 4 months from one electrode that recorded both nonsortable spikes (blue) and sortable AP waveforms (red). The waveforms are isolated and averaged from 3- to 9-min recording segments. Vertical bar, 200 mV; horizontal bar, 1 ms. (E) Principal component (PC) analysis of all the waveforms in (D). Dots: center of the PC. Ovals: 2s contour of PC distribution. Colors code the time stamps. Inset: The evolution over time of the centers of the PC for the single-unit waveform. an example, Fig. 3D shows the bimonthly measurements from the most 1 month and was fully repaired in association with vascular remo- electrode that detected typical spike events and sortable AP waveforms deling. As shown in Fig. 4 (A and B) and movie S2, both types of NET with the highest SNR (>30) among all electrodes. The persistently high probes were embedded in capillaries with normal density, morpholo- SNR of the sortable AP waveforms indicated that the firing neuron re- gy, and an intact BBB by 2 months. We measured the blood flow rate mained in close proximity to the electrode for the entire duration of the by line scans (Fig. 4, C and D) (35) and obtained similar values next to chronic recording (33). Combined principal component analysis for all (420 ± 180 mm/s) and away from (450 ± 210 mm/s) the probe (Materials measurements showed largely overlapping clusters with cluster centers’ and Methods), confirming that capillary perfusion immediately next to average position shifts between consecutive measurements equivalent the probe was normal. This is in strong contrast to conventional micro- to 0.88s of the average cluster distribution (Fig. 3E), suggesting that electrodes where continuous BBB leakage was observed in chronic in vivo the waveforms were generated by the same neuron. In addition, we re- imaging at the interface (36). peatedly detected interneuron waveforms from one electrode (fig. S6), We imaged the astrocytes by in vivo staining (Materials and which were rare events (<10%) and served as a high-probability marker Methods) 3.5 months after implantation around a NET-50 probe for tracking the same neuron (34). Furthermore, we observed persistent (Fig.4Eand movieS3).Wenotethatthe imaged segment was un- characteristics in AP waveforms, whereas minor trackable changes intentionally folded during implantation with a sub–100-mmcurva- occurred for all except one electrode (18 of 19, 94.7%; fig. S7). Signif- ture, which set the upper limit of tissue-probe stress for NET icantly, in contrast to previous neuron probes where decreasing am- probes. We observed that the astrocytes near the probe surrounded plitudes were typically observed over time, most of the NET electrodes only capillaries with normal density and morphology (similar to astro- recorded AP waveforms with nonmonotonic changes in peak-valley cytes from the contralateral hemisphere of the same mouse shown in amplitude. The recording evidence strongly indicates that the NET fig. S9). We also performed postmortem histology (Materials and probes form a nondegrading, slowly evolving interface with neurons. Methods) 5 months after implantation and observed normal neuron To directly investigate the nature of the probe-tissue interface, we density (fig. S10A) and resting microglia (Fig. 4F) near the probe. Re- monitored capillaries, astrocytes, and neurons around the NET probes markably, direct contact with the probe did not affect the viability of by in vivo 2P imaging at the implantation sites for up to 3.5 months after neurons, nor did it activate microglia (Fig. 4F) (24). These results show surgery (Materials and Methods). Figure S8 shows the evolution of the that the NET probes have unprecedented biocompatibility as they vasculature, showing that the surgical damage led to minor local leakage maintain normal vascular and cellular structures and elicit no observ- of the blood-brain barrier (BBB) upon implantation, which lasted for at able chronic tissue reactions at the interface. Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 4of9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 4. Imaging and tracking of the cellular and vascular structures at the chronic probe-tissue interface. (A and B) Three-dimensional (3D) reconstruction of vasculatures by in vivo 2P microscopy around NET-50 (A) and NET-10 (B) probes (red) 2 months after implantation, highlighting fully recovered capillary networks (green). Image stack: 0 to 400 mm (A) and 100 to 320 mm (B) below the brain surface. See movie S2 for a full view of (B). (C) 2P image at 200 mm deep marking the position of a capillary (dashed line) for the line scans in (D). (D) Matrix of line scans showing movement of RBCs as dark stripes, the slope of which gives the blood flow speed. (E)Projection of in vivo 2P images at 210 to 250 mm below the brain surface at 3.5 months after implantation showing normal astrocytes and capillaries. The bright “z”-shaped object is a folded NET-50 probe. The capillaries are visible as dark lines. Right: Zoom-in view of the dashed area. See movie S3 for the full image stack 125 to 360 mm below the brain surface. (F)Projectionof confocal micrographs of an immunochemically labeled cross-sectional slice (30 mm thick, 5 months after implantation). False-color code: orange, NeuN, labeling neuron nuclei; green, lba-1 labeling microglia. White arrows denote microglia soma. Orange arrows denote neurons in contact with the NET probe. (G and H) 3D reconstruction of in vivo 2P images of neurons (yellow) in Thy1-YFP mice surrounding a NET-50 probe (G) and two NET-10 probes (H) 2 and 2.5 months after implantation, respectively. The probes are in red and denoted by arrows. Imaging depth: (G) 130 to 330 mm below the brain surface, (H) 110 to 260 mm below the brain surface. (I and J) Representative 2P images from the same regions in (G) and (H), respectively, showing that neurons are repeatedly identified at different times after implantation. Red dashed lines mark the edge of the probes. Arrows and dashed circles highlight the current and previous locations of neurons, respectively. See movies S4 and S5 for the full image stack. All scale bars, 50 mm. Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 5of9 | SCIENCE ADVANCES RESEARCH ARTICLE To reveal the evolution of the neural interface, we repeatedly imaged cal leakage that results in signal attenuation. However, the possible wa- neurons in the vicinity of both types of NET probes up to 3 months after ter absorption–induced leakage did not affect our long-term recording implantation and compared their locations relative to a few representa- performance, mostly because it did not induce structural damage of the tive neurons and capillaries as position markers (movies S4 and S5 and NET as shown above, likely due to the improved adhesion between Fig. 4, G to J). We observed that all neurons initially imaged near the layers and reduced stress. Furthermore, NET probes were soaked for probe were identified in later imaging sessions, which further proved several days in phosphate-buffered saline (PBS) and other water solu- that the NET probes did not induce chronic neuronal loss. Further- tions before implantation, which preconditioned the SU-8 film to a more, although most neurons were relatively stationary, some show saturated water absorption of 3.3% (40), such that no signal attenuation slow migrations (up to ca. 10 mm) over the course of a few weeks was observed after implantation. Consistent with no structural deterio- (Fig. 4, I and J). Notably, we observed no preference in their migration ration, electrochemical impedance spectroscopy (EIS) showed a high trajectories for moving toward or away from the NET probes. impedance, moderately capacitive circuit over a large frequency range (fig. S11), and little cross-talk was observed from adjacent electrodes (fig. S12). DISCUSSION A very recent study reported stable long-term recording in mice The ultraflexibility of the NET probe prohibits conveniently advancing using mesh electrodes on a similar device platform (31). The con- to and recording from more than one brain region using schemes simi- sistency between their study and ours strongly supports the validity lar to microdrives that are widely applied in silicon probes and tetrodes. of using ultraflexible devices, particularly polymer-based devices with Instead, recording from deeper brain regions or from different brain an overall thickness of about 1 mm, for long-term neural activity map- tissue depths can be realized by implanting NET probes patterned with ping. Here, we further reduced both the device width and the surgical more electrodes spanning a larger depth, especially given the un- footprint to cellular dimensions, such that no persistent glial scar was precedented tissue compatibility demonstrated here. Accommodating observed near the implants in our study. In addition, we characterized more electrodes on one probe does not necessarily increase the probe’s the interface not only by postmortem histology, which gave a dimension, as we can further increase the fabrication resolution by “snapshot” assessment, but also by repeated in vivo imaging of thick using electron beam lithography techniques (37). tissue stacks, which unambiguously revealed the evolution of vascular We have thoroughly examined our full fabrication process and and cellular networks around the implanted NET probes. identified that the loss of yield is mainly due to fabrication defects. Combining the results from chronic recording and in vivo imag- Our fabrication involves seven photolithography steps using a manual ing, we gain a comprehensive understanding of the neural interface mask aligner and three metal deposition steps, which makes it difficult formed by the NET probes. In vivo imaging and histology studies re- to completely avoid microparticles and scratches. Furthermore, our vealed a chronic tissue response free interface, which provides the design has narrow metal lines (the interconnects, 2 to 3 mmwide) rou- biological foundation for nondegrading recording performance. More- ted over 5 mm, which is vulnerable to microdefects. We expect that over, because the interface is dynamically evolving, which involves re- one can significantly improve the fabrication yield by using automated modeling of vasculature and other changes in the microbiological photolithography tools that can eliminate most of the defect sources. environment, both the neurons and the NET probes are not complete- The NET probes can be readily fabricated in linear arrays, similar to ly stationary over a time frame of a few months. Correspondingly, we theconventionalsilicon probes.We estimatethat wecan deliverupto observed neuron migrations at a few micrometers per month, which 12 NET probes within 40 min on the basis of the demonstrated average led to slowly evolving waveforms in chronic recordings that could be delivery time of 3 min per probe. The closest interprobe spacing we have reliably tracked by measurements twice a month. achieved is about 200 mm, as showninFig.2G. Theaccuracy ininter- We demonstrated that the NET probes form reliable, glial scar–free probe space is currently determined by the accuracy in the initial integration with the living brain by chronic recording, in vivo 2P im- positioning of the NET probe (under a stereomicroscope) on the brain aging, and postmortem characterization of the tissue-probe interface. surface before delivery, which has an uncertainty of up to 30 mm. The unprecedented chronic reliability and stability are expected to The NET probes performed stably in mice for at least 4 months, as fundamentally advance both basic and applied neuroscience, as well evidenced by the intact device structure (fig. S10) and the stable imped- as lead to substantial improvement in the brain-machine interface ance and recording performance (Fig. 3), which are sufficient for most which can be applied to neuroprosthetics (41). Furthermore, the sub- scientific studies and warrant further research on their performance in cellular dimension probes provide new opportunities for high-density longer-term implantation (on the order of years). We attribute the sta- electrical recording by overcoming current physical limitations (42). ble performance of NETs to their unconventional dimensions and de- sign, which can be explained by the following aspects: (i) The most prevalent failure modes in conventional devices are structural degrada- MATERIALS AND METHODS tions, such as cracking, blistering, and delamination (38), none of which NET brain probe fabrication and preparation were observed in chronically implanted NETs (fig. S10, B and C). This The NET brain probes were fabricated using specialized fabrication can be attributed to the fact that the ultrathin thickness of NETs (1 mm) methods similar to those previously reported (20, 43). The multilayer greatly reduces the strain on the device upon deformation, which probes were fabricated using photolithography on a nickel metal release prevents the formation of structural defects. (ii) Another major failure layer deposited on a silicon substrate (900 nm SiO , n-type 0.005 V·cm, mode in conventional polymer implants is delamination due to insuf- University Wafer). An SU-8 photoresist (SU-8 2000.5, MicroChem ficient bonding at interfaces (39). In NET probes, the SU-8 insulating Corp.), which offers excellent tensile strength, ease of fabrication, and dem- layer adheres mainly with another SU-8 layer by thermal curing and onstrated durability in ultrathin structures (fig. S10) (20, 23, 43), was cross-linking, which eliminated this failure mode. (iii) Most polymers used to construct the insulating layers. We used the minimum thick- absorb water under physiological conditions, which may lead to electri- ness of the dielectric layer necessary for preventing more than 1% signal Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 6of9 | SCIENCE ADVANCES RESEARCH ARTICLE attenuation through the capacitive coupling between the interconnects probe reached the desired depth, the shuttle device was retracted, and the conductive medium surrounding the probe, which was and the neural probe was released and left embedded in the brain determined to be about 500 nm for our probe geometry and material. tissue. Because the volume of both the shuttle device and the neural Platinum or gold was used for electrodes (size: 30 mm×30 mmforNET- probe was minimized to reduce tissue damage, the delivery process 50 and 10 mm×20 mm for NET-10) and interconnects, respectively, was delicate. A few experimental details are important to the success both with a thickness of 100 nm. The different sizes and materials used of the delivery. First, the anchor post of the shuttle device needs to be for the electrodes contributed to most of the variation in impedance, as microfabricated precisely according to the well-defined cylindrical shown in Fig. 3A. After fabrication, a 33-pin flexible flat connectors shape as designed, so that it can fit into the engaging hole on the probe, (FFC) (series 502598, Molex) was mounted on the matching contact can have sufficient mechanical strength for the delivery, and can be pads on the Si substrate. The implantable section of the probe was then retracted out of the brain without pulling the probe out with it. Second, soaked in nickel etchant (TFB, Transene Co. Inc.) for 2 to 4 hours at theaxial directionofthe shuttledeviceneeds to be alignedwiththe 25°C to release the free-standing portion of the probe, while the contact insertion direction to minimize buckling. Third, ACSF needs to be region remained attached to the substrate. The substrate was cleaved to added to the brain surface as the shuttle device drags the probe into the desired length before implantation. the brain to float the remaining flexible segment and to minimize fric- tion between the probe and the brain surface. By controlling the Shuttle device fabrication and assembly engaging location and the insertion depth, we delivered NET probes A straight segment of a carbon fiber or tungsten wire was attached to a to specific regions of interest. Multiple-probe delivery was achieved by stainless steel microneedle (prod# 13561–10, Ted Pella Inc.) for con- repeating the same process sequentially. venient handling. It was then cut to the designed length (2 to 3 mm) using FIB. An anchor post was micromilled at the tip of the shuttle de- 2P imaging vice using FIB to shape a well-defined micropost (3 mm in diameter 2P imaging was performed using a laser scanning microscope (Prairie and 5 mm in height, fig. S4). Technology) equipped with a 20× water immersion objective (numer- ical aperture, 1.0; Zeiss) and a Ti:sapphire excitation laser (Mai Tai Animals and surgery DeepSee, Spectra-Physics) from acutetoupto3.5 months afterim- Wild-type male mice (C57BJ/6, 8 weeks old, Taconic) and male trans- plantation. The laser was tuned to 810 to 910 nm for 2P excitation genic strain [B6.Cg-Tg(Thy1-YFP)16Jrs/J, to which yellow fluorescent (power,3.0 to 50 mW;dwell time,4.0 to 6.0 ms). Fluorescence emis- proteins were expressed in neurons; The Jackson Laboratory] were sions were detected simultaneously by two standard photomultiplier used in the experiments. Mice were housed at the Animal Resources tubes with a 595/50-nm filter (Semrock) for “red” fluorescence emis- Center at the University of Texas (UT) at Austin (at a 12-hour light/ sion and a 525/70-nm filter (Semrock) for “green” fluorescence emis- dark cycle at 22°C, with food and water ad libitum). sion. Mice were anesthetized using isoflurane (3% for induction and Mice were anesthetized using isoflurane (3% for induction and 1.5% during experiment) in medical-grade oxygen to maintain full im- maintained at 1 to 2%) in medical-grade oxygen. The skull was exposed mobility duringimaging andplacedinaframethatstabilizedthe head and prepared by scalping the crown and removing the fascia and then on the microscope stage. For 2P imaging on capillaries, anesthetized was scored with the tip of a scalpel blade. A 3-mm × 3-mm square animals were given fluorescein isothiocyanate (FITC)–dextran (0.1 ml, craniotomy was performed with a surgical drill over the somato- 5% w/v, Sigma-Aldrich) retro-orbitally to label blood vessels before sensory cortex. Dura mater was carefully removed to facilitate the de- imaging. For 2P imaging on astrocytes, anesthetized animals were livery. After NET probe implantation (described below), the remaining given sulforhodamine 101 (44) (0.1 ml, Sigma-Aldrich) and Optison flexible segment of the NET probe, which connected the bonding pad (GE Healthcare, 0.1 ml) retro-orbitally. Immediately following the on the substrate with the electrodes inside the brain, was routed to the injection, focused ultrasound with a center frequency of 2.25 MHz edge of the cranial opening. The exposed brain was then protected by in the tone burst mode (rate, 20 Hz; burst duration, 10 ms; duty artificial cerebrospinal fluid (ACSF) and coverslip #1 (Fisher Scientific) cycle, 20%) was applied to the regions of interest for 45 s to tempora- fit into the cranial opening. The space between the coverslip and the rily break the BBB (45). Animals were imaged 2 hours after ultra- remaining skull was filled with Kwik-Sil adhesive (World Precision sound treatment to ensure sufficient dye diffusion and staining. To Instruments). After the skull was cleaned and dried, a layer of low- facilitate the imaging of the probe-tissue interface beyond superficial viscosity cyanoacrylate was applied over the skull. An initial layer of cortical layers, we doped the probes with sulforhodamine 6G (Sigma- C&B-Metabond (Parkell Inc.) was applied over the cyanoacrylate and Aldrich) in the insulating layers and delivered them at about 45° with the Kwik-Sil adhesive. A second layer of Metabond was used to cement respect to the skull. the coverslip and the NET carrier chip to the skull. All procedures complied with the National Institutes of Health guidelines for the care Line scan and blood flow speed and use of laboratory animals and were approved by the UT Institutional We inferred the blood flow speed from the motion of red blood cells Animal Care and Use Committee. (RBCs) shown as stripes in line-scan measurements, that is, repetitive scans of the laser along the central axis of a capillary. The animal was Neural probe delivery given FITC-dextran (0.1 ml, 5% w/v, Sigma-Aldrich) retro-orbitally before In typical procedures, a flexible neural probe was placed on the imaging. After obtaining a 2P image of capillaries at the desired depth, brain surface where dura mater was removed. The shuttle device was we identified a capillary of interest and electromechanically oriented vertically mounted on a micromanipulator (MP-285, Sutter Instrument) the direction of the scan along it. We typically scanned a distance of and positioned atop the engaging hole at the end of the probe. As 30 to 40 mm with 128 pixels per line, repeating 128 to 256 times at the shuttle device traveled downward, the anchor post entered the hole 1 to 2 ms per scan. We imaged nine capillaries in the vicinity of a and pulled the neural probe into the brain tissue. Once the neural NET-50 probe and six capillaries from the contralateral hemisphere Luan et al. Sci. Adv. 2017; 3 : e1601966 15 February 2017 7of9 | SCIENCE ADVANCES RESEARCH ARTICLE table S1. Animal- and probe-specific recording performance over time. of the same mouse. The blood flow speed was 420 ± 180 mm/s near movie S1. A typical implantation procedure of the NET probe. the probe, similar to 450 ± 210 mm/s away from the probe. movie S2. The NET-vasculature integration 2 months after implantation. movie S3. In vivo 2P images of inactive astrocytes and intact capillary network around a folded Histological sample preparation segment of a NET-50 probe 3 months after implantation. movie S4. In vivo 2P images of neurons and two NET-10 probes 1.5 and 2.5 months after Mice were given lethal intraperitoneal injections of 0.15 ml of ketamine implantation. mixed with xylazine [xylazine (10 mg/ml) in ketamine (90 mg/ml)] and movie S5. 3D reconstruction of in vivo 2P images of neurons and two NET-10 probes then perfused intracardially with oxygenated, cold (∼4°C) modified 1.5 months after implantation (the same images as in movie S4, left). ACSF (2.5 mM KCl, 1.25 mM NaH PO ,25mMNaHCO ,0.5 mM 2 4 3 References (46, 47) CaCl , 7 mM MgCl , 7 mM dextrose, 205.5 mM sucrose, 1.3 mM as- 2 2 corbic acid, and 3.7 mM pyruvate) followed by 4% paraformaldehyde in 0.02 M PBS. Brains were cryoprotected in a 30% sucrose/4% para- REFERENCES AND NOTES formaldehyde solution overnight. Tissue was sectioned into 20- to 1. C. M. Gray, P. E. Maldonado, M. Wilson, B. 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Science Advances – Pubmed Central
Published: Feb 15, 2017