Long-term in vivo monitoring of gliotic sheathing of ultrathin entropic coated brain microprobes with fiber-based optical coherence tomography
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Journal of Neural Engineering
PAPER • OPEN ACCESS
Long-term in vivo monitoring of gliotic sheathing of ultrathin entropic
coated brain microprobes with fiber-based optical coherence
tomography
To cite this article: Ian Dryg et al 2021 J. Neural Eng. 18 045002
View the article online for updates and enhancements.
This content was downloaded from IP address 132.230.59.55 on 14/04/2021 at 08:47
J. Neural Eng. 18 (2021) 045002 https://doi.org/10.1088/1741-2552/abebc2
Journal of Neural Engineering
PAPER
Long-term in vivo monitoring of gliotic sheathing of ultrathin
OPEN ACCESS
entropic coated brain microprobes with fiber-based optical
RECEIVED
22 October 2020 coherence tomography
REVISED
14 February 2021 Ian Dryg1,2,6, Yijing Xie3,6,7, Michael Bergmann4, Gerald Urban4, William Shain1,2
ACCEPTED FOR PUBLICATION
3 March 2021
and Ulrich G Hofmann3,5,∗
1
Department of Bioengineering, University of Washington, Seattle, WA, United States of America
PUBLISHED 2
23 March 2021 Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, United States of America
3
Section for Neuroelectronic Systems, Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany
4
Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany
Original content from 5
Faculty of Medicine, University of Freiburg, Freiburg, Germany
this work may be used 6
under the terms of the These authors have equal contributions.
7
Creative Commons Current address: School of Biomedical Engineering and Medical Imaging, King’s College London, London, UK.
∗
Attribution 4.0 licence. Author to whom any correspondence should be addressed.
Any further distribution
of this work must E-mail: ulrich.hofmann@coregen.uni-freiburg.de
maintain attribution to
the author(s) and the title Keywords: neural interfaces, foreign body reaction, optical coherence tomography, immunohistochemistry, plasma coating,
of the work, journal biocompatibility
citation and DOI.
Supplementary material for this article is available online
Abstract
Objective. Microfabricated neuroprosthetic devices have made possible important observations on
neuron activity; however, long-term high-fidelity recording performance of these devices has yet to
be realized. Tissue-device interactions appear to be a primary source of lost recording
performance. The current state of the art for visualizing the tissue response surrounding brain
implants in animals is immunohistochemistry + confocal microscopy, which is mainly performed
after sacrificing the animal. Monitoring the tissue response as it develops could reveal important
features of the response which may inform improvements in electrode design. Approach. Optical
coherence tomography (OCT), an imaging technique commonly used in ophthalmology, has
already been adapted for imaging of brain tissue. Here, we use OCT to achieve real-time, in vivo
monitoring of the tissue response surrounding chronically implanted neural devices. The
employed tissue-response-provoking implants are coated with a plasma-deposited nanofilm, which
has been demonstrated as a biocompatible and anti-inflammatory interface for indwelling devices.
We evaluate the method by comparing the OCT results to traditional histology qualitatively and
quantitatively. Main results. The differences in OCT signal across the implantation period between
the plasma group and the control reveal that the plasma-type coating of otherwise rigid brain
probes (glass) only slightly improve the glial encapsulation in the brain parenchyma indicating that
geometrical or mechanical influences are dominating the encapsulation process. Significance. Our
approach can long-term monitor and compare the tissue-response to chronically-implanted neural
probes with and withour plasma coating in living animal models. Our findings provide valuable
insigh to the well acknowledged yet not solved challenge.
1. Introduction (Fanselow et al 2000, Rouse et al 2011, Tooker et al
2014, Pinnell et al 2016, 2018). These neural implants
Implantable neural microelectrodes are developed are designed to stay within the nervous system over
to deliver therapeutic electrical stimulus for treat- a long period up to years (Krüger 2010, Prasad
ing neural degenerative diseases, and to collect et al 2012, Jorfi et al 2015, Nolta et al 2015). How-
electrophysiology signals from surrounding neurons ever, the performance of the electrode in terms of
© 2021 The Author(s). Published by IOP Publishing Ltd
J. Neural Eng. 18 (2021) 045002 I Dryg et al
signal transferring efficacy and measurement signal- vivo whole rat brain trajectories (Xie et al 2013) and
to-noise ratio deteriorate over time due to the inev- flexible implants (Xie et al 2014), and elucidated
itable foreign body reaction which eventually forms the bases of its contrast’s origins (Xie et al 2017).
a dense glial sheath to encapsulate the implant, isol- However, we observed OCT signal deterioration over
ating it from the neural environment (Szarowski et al time along the implantation duration presumably
2003, Kim et al 2004, Polikov et al 2005, Williams et al due the gliotic encapsulation around the OCT detec-
2007, Ward et al 2009, Barrese et al 2013). tion probe itself. In this study, we first sought to com-
To realize a long-term reliable neuro-electrode pare the ability of fiber-based OCT with traditional
interface, it seems essential to minimize the foreign IHC in observing the glial scar around another rigid
body response (Winslow and Tresco 2010, Nguyen implanted device (i.e. a silicon probe). Second, we
et al 2014, Potter et al 2014, Sohal et al 2016). To used fiber-based OCT to investigate the foreign-body
this end, many approaches have been proposed and response of surface modifications by plasma depos-
investigated including innovations on bio-compatible ited methane coatings, which have shown promise for
flexible materials (Stieglitz and Meyer 1998, Rousche affecting protein adsorption, reducing cellular adhe-
et al 2001, Takeuchi et al 2004, Rubehn et al 2010, sion, and reducing tissue encapsulation intravascu-
Richter et al 2013, Nguyen et al 2014, Sohal et al 2016, larly in pigs (Bergmann et al 2015, Bergmann 2015).
Böhler et al 2017, Luan et al 2017), decreasing implant
size (Patel et al 2015, 2016, Khilwani et al 2017, Ferro 2. Materials and methods
et al 2018, Yang et al 2019) or surface modifications by
coating the device with anti-inflammatory drugs that Six female Sprague Dawley rats were each implanted
will actively release (Azemi et al 2011, Kolarcik et al with one inactive silicon probe and one OCT probe at
2012, Potter et al 2014, Boehler et al 2017, Eles et al 90◦ to one another as shown in figure 1. To assess glial
2017). scar development over time around silicon probes,
These approaches are often evaluated by analyz- OCT monitoring was performed weekly for up to
ing cellular changes corresponding to the progres- 8 weeks before sacrificing for histological assessment
sion of the foreign body reaction over time following with IHC. To investigate the effect of plasma depos-
implantation, mostly based on postmortem immun- ited CH4 coatings on glial scar development around
ohistochemistry (IHC) (Turner, 1999, Szarowski et al implanted device, the implantable OCT probes were
2003, Biran et al 2007, Ward et al 2009, Lo et al 2018). separated into CH4 -coated group and uncoated con-
The process of the foreign body reaction incorporates trol group. Then, tissue properties were assessed by
a rapid activation of microglial cells immediately fol- measuring the tissue attenuation coefficient of the
lowing the implantation; and as the response develops OCT signal (Xie et al 2013, 2014) and finally by IHC.
up to 3 months, it reaches a chronic phase with com- We chose to apply coating on OCT probes rather
pact astrocytic scar tissue generated to sheathe the than silicon probes as it is up till now unclear, which
implant (Leach 2010, Pflüger et al 2019). Normally, effects the CH4 coating might have on the electrical
a large number of implanted and control animals are properties of probe’s metal recording sites. We fur-
sacrificed to obtain sufficient temporal resolution of ther wanted to investigate (a) if there were detect-
the progression curve as well as to reach a statistical able changes in the tissue surrounding the OCT probe
significance. Furthermore, it is impractical to mon- due to the coatings, (b) whether the coatings would
itor the complete course of progression of the same diminish the tissue foreign-body reaction thus rectify
object using postmortem histology. OCT performance on monitoring the tissue changes
In vivo two-photon microscopy (TPM) has been over time around the silicon microelectrode probes.
adopted for continuous monitoring the progression Due to the light attenuation in tissue eventual differ-
of the biological processes in neural tissue in response ence in tissue response due to the coatings would be
to foreign implants (Kozai et al 2016, Eles et al 2017, most detectable closest to the OCT probe.
Wellman and Kozai 2018). It has been demonstrated
that TPM is able to obtain spatiotemporal informa- 2.1. Implantable probes: optical coherence
tion of the dynamic cellular response to the implanted tomography fiber probe and silicon microelectrode
electrodes up to 12 weeks (Kozai et al 2016). How- probe
ever, the imaging depth of TPM is limited within The customized OCT fiber probe (SM800-
hundreds of micrometers (e.g. 200 µm), which only CANNULA, Thorlabs) consists of a ceramic fiber
allows investigation of immune reactions very close to connector ferrule (CF126-10, Thorlabs) with 2.5 mm
the brain’s surface. diameter and 10.5 mm length and an 8 mm long
Here we report our approach using fiber-based single mode fiber (SMF) (ø 125 µm, SM800-5.6-125,
optical coherence tomography (OCT) to monitor Thorlabs) with clean cleaved distal end that is per-
the development of the glial scar in vivo over the pendicular to the fiber’s longitudinal axis. The single
course of the implantation. We previously demon- mode fiber is implanted into brain tissue to enable
strated the use of fiber-based OCT to gain insight monitoring foreign body reaction in situ, while the
as a minimally-invasive imaging modality during in ferrule remains exposed and is connected with the
2
J. Neural Eng. 18 (2021) 045002 I Dryg et al
Figure 1. Surgical setup. (a), (b) To detect the silicon probe with the OCT signal, probes had to be angled as close to 90◦ from one
another as possible, maximizing reflectance back into the OCT probe. (c) Top view of the surgical setup. A burr hole line was
drilled to allow implantation of probes into brain tissue. The OCT probe and silicon probe were pre-aligned before advancing
into brain tissue. The silicon probe was advanced first, followed by the OCT probe until the silicon probe could be detected in the
OCT signal. (d) OCT signal during surgery after both probes are aligned and implanted, showing the reflectance of the implanted
silicon probe.
SMF cable of the OCT system each time the OCT under micromanipulator control to a strict perpen-
measurement is conducted. The silicon-based rigid dicular implant geometry into each animal. To study
microelectrode probe is chosen in this study as it has the effect of CH4 plasma coating (Bergmann et al
a wide range of applications in neuroscience. Thus, a 2015, Bergmann 2015) on the foreign body reaction,
spatiotemporal in situ assessment on the foreign body three of the rats were implanted with CH4 plasma
reaction against the chronically implanted silicon coated OCT probes. The remaining OCT probes and
probe would be of great interest to neuroscientists. all of the silicon probes were uncoated. G∗ Power
The silicon probe used here is 50 µm thick, 140 µm (Faul et al 2007) was used to perform post-hoc power
wide and 10 mm long. Both OCT fiber probes and analyses, outlined in table S1.
silicon microelectrode probes are immersed in 70% The surgical procedure was performed as previ-
ethanol for 20 min for disinfection, and rinsed thor- ously described (Richter et al 2013). In brief, rats were
oughly with sterile saline (0.9% Sodium Chloride) anesthetized (induction: 5% isoflurane in 2 l min−1
before implantation. O2 ; maintenance: 0.5%–3% isoflurane in 2 l min−1
O2 ), the surgical area was shaved, and rats were placed
2.2. Plasma deposition of CH4 coating on glass on a water-circulating heating pad to maintain a body
OCT probes temperature of 35 ◦ C. The rats were fixed into a ste-
Using methane as the starting material, silicon probes reotactic frame (Kopf Instruments), and eyes were
were plasma coated as previously described (Ledernez covered with ophthalmic ointment. The surgical area
2011, Bergmann et al 2015). Briefly, a magnetron was sterilized using alternating wipes of polyvidone-
enhanced plasma polymerization process was con- iodine and alcohol. A midline incision was made
ducted in a system from the company Shinko Seiki, along the scalp, and the skin was pulled aside to
with two parallel titanium electrodes separated by expose the skull’s surface. Bregma was identified and
10 cm. Samples were rotated between the electrodes used as a landmark coordinate (0, 0 mm). Bone
to ensure good homogeneity of the resulting plasma screws were placed into the skull anterior to bregma,
films. The plasma chamber was evacuated down to a posterior and lateral (left) to bregma, and posterior
pressure of 0.1 Pa to avoid cross-contamination, then to lambda, to better secure a headcap with dental
coated at a pressure of 5 Pa using a power of 45 W. acrylic later. A burr hole line 9 mm long and 1 mm
wide was drilled from 1 mm posterior and 3 mm
2.3. Implant surgery lateral (right) to 10 mm posterior and 3 mm lateral
All experiments in this study were performed with (right) of bregma. Then, using two different arms on
approval from the locally responsible Animal Welfare the stereotactic frame, the implantable OCT imaging
Committee with the Regierungspräsidium Freiburg probe and the silicon probe were positioned above
in accordance with the guidelines of the European the burr hole line, each angled 45◦ up from hori-
Union Directive 2010/63/UE under permit G13/51. zontal, and angled 90◦ from one another (figure 1).
We used eight adult female Sprague Dawley rats The OCT probe was connected to the OCT imaging
(Charles River, Germany) weighing 280–320 g, system, and the OCT probe and silicon probe were
among which six rats (three rats in each group) aligned laterally, pointing the OCT probe at the sil-
underwent successful probe implantation and were icon probe ∼1 mm away, until the strongest reflect-
used for OCT recording and histology analysis. One ance signal from the silicon probe was achieved in the
silicon probe and one OCT probe were implanted OCT signal. This process was essentially a practice run
3J. Neural Eng. 18 (2021) 045002 I Dryg et al
in air, creating the correct alignment and positioning The single mode fiber (5.6 µm diameter core,
of the probes for best imaging signal when implanted numerical aperture = 0.12) has an acceptance angle
into tissue. Once correctly aligned in the medio- of 6.9◦ . Thus, the back scattered light collected by
lateral direction, the probes were separated along the the OCT probes was within a conical shape starting
antero-posterior direction, and the silicon probe was at the OCT probe tip and diverging at an angle of
advanced into brain tissue ∼10 mm, maintaining 6.9◦ . To successfully image the tissue response around
the same angle and lateral positioning as during the the silicon probe, the light projecting from the OCT
alignment in air. Then, the OCT probe was slowly probe must shine on the silicon probe penetrating
advanced into brain tissue, until the OCT reflectance the tissue around it. It was possible to see the silicon
signal from the silicon probe was detected ∼1–2 mm probe as a strong reflectance signal in the OCT signal
away from the OCT probe tip. The OCT probe was (see figure 1(d) about 0.75 mm deep into the tissue),
advanced until the distance between the OCT probe and this reflectance signal was used to confirm that
tip and the silicon probe was 1 mm, as measured by the OCT probe was pointed correctly at the silicon
the OCT signal. The entire burr hole line including probe. However, this only worked if the OCT probe
the entry points of the probes were covered in gelfoam was oriented perpendicular to the silicon probe. If ori-
(Pfizer) wetted with sterile saline, then with silicone ented other than 90◦ , the reflectance signal from the
elastomer (Kwik-Sil, World Precision Instruments), silicon probe was diminished (figure 1(a)). Prior to
and finally sealed with dental acrylic. Dental acrylic OCT and silicon probe implantation, the two probes
was sequentially applied in several layers, securing the were oriented in air above the animal to ensure that
implanted probes in place, and building the dental the probes were lined up correctly, as confirmed by
acrylic headcap to cover the wound of the animal. The the strong reflectance signal from the silicon probe
ferrule interfacing between the implanted OCT probe in the OCT signal. Once probes were correctly lined
and the fiber optic cable leading to the OCT ima- up, implantation surgery proceeded. After both OCT
ging system was left uncovered from dental acrylic, and silicon probes had been advanced into tissue, it
enabling access for connection to the OCT imaging could be verified that the OCT probe was pointed
system throughout the study. Carprofen (4 mg kg−1 ) directly at the silicon probe using the reflectance sig-
was administered subcutaneously for 5 days post- nal from the silicon probe. An example of this veri-
surgery to manage pain. The animals were housed fication is shown in the raw OCT A-scans data in
separately with enrichment and daily inspection. figure 1(d). OCT recording is performed live and new
data is streamed into the visualization window con-
2.4. Optical coherence tomography tinuously. Figure 1(d) shows an approx. 1 s recod-
The OCT imaging system used in this study is ing of OCT A-scans of this live streaming (referred
identical to that which is previously described (Xie as ‘snapshot’ in this report), where the x-axis is time
et al 2013, 2014). The fiber-based spectral radar OCT and the y-axis is distance into tissue with the top of the
system utilizes a superluminescent diode with cen- image is the tip of the OCT imaging probe, and tissue
ter wavelength at 840 nm as light source. The sys- depth increases going down the y-axis. The strength
tem axial resolution is approximately 14.5 µm meas- of the OCT signal is represented as brighter points
ured in air. Briefly, we used OCT (Fercher et al 1993) in the image. The reflectance signal from the silicon
to monitor the development of foreign body cap- probe can be seen as the bright line about 0.7 mm
sule formation around implanted OCT fiber probes deep into tissue in figure 1(d). OCT A-scans data
and silicon probes in brain tissue. The implanted from all animals throughout the implantation period
fiber transmits incident light into brain tissue and is also represented using these snapshots in figure
collects light that scatters back into the fiber. The S1 (available online at stacks.iop.org/JNE/18/045002/
intrinsic optical properties of the tissue are encoded mmedia). In order to analyze the effect of coating on
by the interference pattern created by the incident and OCT probe’s characteristics with regard to the output
backscattered light, which is detected by a spectro- power of light and its numerical aperture, we meas-
meter to construct A-scan (one-dimensional depth ured the output beam profile and power of the coated
scan) signals that present backscattered light intens- and uncoated OCT probes using a power meter (400–
ity as a function of depth. As our OCT utilizes a 1100 nm, PM160, Thorlabs).
low-coherence superluminescent light source in the Implanted OCT fibers were either coated with
near infrared portion of the spectrum (840 nm), con- plasma-deposited CH4 or left uncoated. After a CH4 -
trast in the OCT signal predominantly depends on coated or uncoated fiber had been implanted and
the attenuation (optical density of tissue and cells) fixed in place within the dental cement headcap, the
and the dichroitic properties (like myelin fibers) of the fiber optic cable connecting the implanted fiber to the
illuminated tissue (Kut et al 2015). Both sending and OCT imaging system could be disconnected, allow-
receiving optics are defined by a cleaved single mode ing the animal to move around freely. After discon-
fiber (0.12 NA, SM800-5.6-125, Thorlabs) achieving necting the implanted OCT probe, the ferrule was
extremely high localization of beams and thus making wrapped in parafilm for protection. Before each ima-
perfect arrangement an important requirement. ging session, the parafilm was removed and the ferrule
4J. Neural Eng. 18 (2021) 045002 I Dryg et al
was cleaned using Fiber Connector Cleaning Solu- a 3D representation of the fluorescence data. Finally,
tion (FCS3, Thorlabs). The implanted OCT probe all optical sections were compiled together into one
was then connected to the OCT system via the fiber image by taking a maximum projection. All images
optic cable and in vivo imaging was performed. If a were captured using the same settings across fluores-
poor signal was observed, the fiber optic cable was cence channels.
removed, the ferrule re-cleaned, and the OCT system To assess the effects of CH4 plasma coating on
re-connected to try again. Rats were not anesthetized the foreign body reaction, the fluorescence of glial
during OCT imaging, but only data recorded while cell markers around implants were examined. Ana-
the animal was motionless were used for analysis, as lysis of the glial cell responses surrounding implants
movement of the animal can disrupt the OCT signal was achieved using a custom MATLAB script that
quality. To create an OCT signal profile, imaging data measured the average fluorescence intensity of each
over an entire second of motionless collection was channel versus distance from the implant in 10×
averaged together and plotted as OCT signal intensity images. Fluorescence profiles from all animals in each
vs distance from the tip of the OCT probe. group were averaged together, normalized to control
fluorescence (no primary antibody) and subjected
2.5. Tissue histology to statistical analysis (student’s t-test). Comparis-
After 8 weeks, rats were given an intraperitoneal injec- ons between coated and uncoated groups were per-
tion of ketamine/xylazine and sacrificed by cardiac formed, as well as coated vs control, and uncoated
perfusion with PBS to clear the blood, and then vs control comparisons. Control data was gathered
4% paraformaldehyde to fix the tissue. Rat heads by averaging the fluorescence intensity of a field far
were removed and post-fixed in 4% paraformalde- (>2 mm) from implants but at the same tissue depth.
hyde overnight, then washed in phosphate buffered Statistical comparisons were performed every 5 µms
saline (PBS) and stored in EPES-buffered Hank’s from the implant edge. As an additional assessment of
Solution (HBHS) with sodium azide prior to brain the effect of the CH4 plasma coating, the OCT signal’s
removal. Because we wanted to compare the OCT sig- attenuation factor was calculated for each probe.
nals to tissue histology, we aimed to maintain both the To compare the OCT imaging signal to the IHC,
implanted OCT imaging probe and silicon probe in 30× image scans of tissue between the OCT probe
situ within one slice to prevent distortion effect on the and the silicon probe were collected. In those images,
surrounding tissue due to probe extraction proced- the shape of the light collected and imaged by the
ures, as demonstrated previously (Woolley et al 2011, OCT probe was recreated. As aforementioned, the
2013). To do this, headcaps were carefully drilled into light collected by OCT probe was within a 6.9◦ conical
using a surgical drill, and implants were cut at the shape space. The IHC images were analyzed by meas-
entry point through the skull to maintain implant uring fluorescence profiles from the tip of the OCT
positioning within brain tissue. Tissue blocks were probe outwards in a conical shape, rotating the angle
carefully aligned for sagittal slicing, keeping both of the profiles to cover the whole area of the cone,
probes along the plane of slicing. Using a vibratome, and then averaging those profiles together to create
tissue blocks were supported with agar gel and sliced one profile. In this way, the OCT imaging signal could
at 300 µm thickness to capture both probes within a be compared to the analogous fluorescence profile
single slice. Slices were immunolabeled using primary from the same tissue that the OCT system imaged
antibodies GFAP (1:1000 dilution, rat IgG2a, Invit- (figure 2).
rogen cat# 13-0300) to label astrocytes, Iba1 (1:250
dilution, rabbit IgG, Wako cat# 016-20001) to label
microglia, NeuroTrace 640/660 (1:250 dilution, Invit- 3. Results and discussions
rogen cat# N21483) Nissl stain to label neuronal cell
bodies, and Hoechst 33342 (1:1000 dilution, Ther- 3.1. OCT probe and recording
moFisher) to label cell nuclei. Secondary antibodies From the test experiments aligning the OCT probe
goat anti-rat AlexaFluor 488 (1:200) and goat anti- and the silicon probe mentioned in section 2.4, the
rabbit AlexaFluor 594 (1:200) were used to target OCT signal reached a maximum when its fiber probe
GFAP and Iba1 primaries, respectively. Slices were was positioned perpendicular to the silicon probe. An
mounted in Fluoromount G and cover slips were OCT signal above 50% of maximum was recorded
sealed with nail polish. Slides were imaged at 10× and between 81◦ and 99◦ (figure 1(a)). We also found
30× using an Olympus spinning disc fluorescence there was no impact on OCT signal in terms of intens-
microscope, scanning across multiple imaging fields ity and divergence angle from the plasma coating.
to image the entire area of interest. Optical sections The OCT signal of each individual rat at their
were taken every 0.3 µm throughout the thickness of respective sampling points (days post implantation
the slice to gather fluorescence data from the whole surgery) over the entire course of implant duration
tissue slice of 300 µm thick. Background subtrac- is shown in figure S1. The implanted silicon probe
tion was performed, and a custom MATLAB script was visible in the OCT signal for all rats at the
stitched the scanned image stacks together to create day of implantation, and the distance of the silicon
5J. Neural Eng. 18 (2021) 045002 I Dryg et al
Figure 2. Qualitative comparisons between IHC (a), (b) and OCT signal (c) for one uncoated sample (left) and one CH4 plasma
coated sample (right) that showed satisfactory OCT signal until the final day of imaging. (a) 10× scans show wide scans
encompassing both OCT and silicon probes or probe tracts which were captured within the tissue slice (scalebar: 500 µm). (b)
30× scans of the tissue between the two probes that had been imaged by OCT (illustrated by white beam) allows comparison
between OCT signal and IHC profiles. Implants are outlined by dotted lines (scale bar: 100 µm). (c) OCT signal profile plotted
below fluorescence profiles from the same tissue that had been imaged by OCT. The x-axis spans from the OCT imaging probe
tip, through the tissue, and ends at the implanted silicon probe. Increases in fluorescence intensity profiles from labeled glial cells
(dotted red line) match with the increase in OCT signal at the location of the silicon probe (solid red line). The two red arrows in
the right panel indicate the respective Iba1 and GFAP activities expanding to ∼1 mm distance from the OCT probe tip.
probe from the tip of the OCT probe was approx- similar manner as reflected in the attenuation value
imately 1 mm for all rats as shown in the OCT of the OCT signal (figures S1 and S3). The atten-
recordings. The implantation procedure was tracked uation factor of OCT signal represents how much
by the OCT. It provided a direct evident of a suc- light attenuates when propagate within a medium. In
cessful implantation in which the final position of biological tissue, the higher attenuation factor often
the silicon was confirmed with the depth distance links to tissue with compact structure, which sup-
and intensity of the silicon probe in OCT signal. As ports the findings here that compact gliotic sheath-
foreign body reaction developed across the implant ing was formed around the OCT probe regardless of
period up to 56 days post-surgery, surprisingly there coating. Moreover, the OCT signal of silicon probes
was no marked difference in OCT signal between was found to become more prominent post implant-
rats with coated and uncoated OCT probe. The tis- ation surgery. We speculate that likewise the sil-
sue developing around the OCT probe progressed in icon probe provoked a foreign body reaction and a
6J. Neural Eng. 18 (2021) 045002 I Dryg et al
dense layer of gliotic scar formed during the implant one animal in figure S2). This may be an important
period, thus it presented increasing high reflectance observation since electrode position relative to nearby
OCT signals. However, this effect demonstrated no neurons greatly affects the signals recorded by that
marked difference in between the two groups in electrode.
comparison.
3.3. Effect of CH4 plasma coating on foreign body
3.2. Comparison of OCT signal to tissue histology reaction
by IHC A qualitative comparison between representative
One goal of this study was to compare OCT signals coated and uncoated samples can be seen in figure 2.
with traditional IHC. At the beginning of the study, These samples were chosen because OCT imaging
the silicon probe was clearly visible in all OCT ima- maintained good quality until the final day in both
ging signals. However, by the end of the implantation of these animals, and the probes or probe tracts were
period, some animals did not have silicon probes vis- well preserved within the tissue slice for IHC in these
ible in the OCT anymore. For these animals, the com- animals. Figure 2(c) shows the fluorescent profiles of
parison between OCT signal on the final day and IHC glial cells along the imaging path, with the OCT probe
was not possible. Two animals for which the silicon at the left end of the x-axis. For this sample, microglia
probe OCT signal was still present on the final day are (iba1) encapsulation was tight around the uncoated
shown in figure 2. It is uncertain why signal was lost OCT probe (figure 2(c)-left) as shown by the peak
for the other animals, but it is likely that the sensit- at the far left of the plot, whereas astrocyte (GFAP)
ive alignment of the two probes shifted, meaning the encapsulation was seemingly minimal. For this coated
light from the OCT probe was not reflecting back off OCT probe (figure 2(c)-right), encapsulation of both
the silicon probe by the end of the study. The vari- microglia (iba1) and astrocytes (GFAP) seemed to
ability in OCT signal over time can be seen in figures extend farther out into tissue as shown by the hump
S1 and S2, where the position and reflectance signal centered around 0.10 mm along the x-axis (marked
from the silicon probe is shown to change over time. with red arrows).
This is likely due to slight changes in positioning of To quantitatively assess the effect of CH4 plasma
one or both of the probes throughout the course of coating on glial encapsulation of the implanted OCT
the study. probes, multiple comparisons were performed: one
Because of the contribution from glial cells in comparing uncoated vs coated OCT probes using the
typical neural implant encapsulation, we assessed attenuation signal from the OCT imaging (figure S3),
iba1 (microglia), GFAP (astrocyte), and cell nuc- and the rest using IHC fluorescence for glial cells sur-
lei (DAPI) fluorescence along the OCT light path rounding the OCT probes (figure 3).
between the two implants using IHC. We hypo- First, OCT signal attenuation revealed no trends
thesized that we would be able to see similarities or significant differences between the uncoated and
in these fluorescence profiles and the OCT ima- coated groups.
ging profiles. When comparing the OCT signal to Second, uncoated vs coated comparisons were
the fluorescence profiles from the tissue histology performed. Uncoated implants were directly com-
images, qualitative similarities were observed. Both pared to coated implants, measuring GFAP (astro-
OCT signal and fluorescence of glial cell markers cytes) and iba1 (microglia) fluorescence vs distance
were increased at the location of the implanted silicon from the probe and performing student t-tests every
probe (figure 2(c), red lines) where the highest degree 5 µms from the edge of the implants. However, there
of glial encapsulation is expected. However, it was dif- were no significant differences between the uncoated
ficult to see any other strong quantitative similarities and coated groups by direct comparison.
between OCT imaging and the glial cell fluorescent Third, both treatment groups were compared
profiles. separately to control tissue (no implant). Specific
The location of the glial scar in fluorescent plots comparisons performed were: uncoated vs control,
matched the location of the implanted silicon probe and coated vs control, measuring GFAP (astrocytes)
in the OCT signal. However, we were unable to find a and iba1 (microglia) fluorescence vs distance from
difference in the extent of glial scarring around the sil- the probe and performing student t-tests every 5 µms
icon probes over time using the OCT signal. From Xie from the edge of the implants. Both coated and
et al (2017), it was determined that OCT image con- uncoated OCT probes showed significantly higher
trast in brain tissue is more likely from fibrous, well- GFAP (figure 3(a)) and iba1 (figure 3(b)) fluor-
ordered, myelin-rich structures such as axons, than escence around the implants compared to control
from other cells such as glia. This helps to explain why tissue (no implant). However, the significant differ-
we were unable to detect changes in gliosis over time ences did not extend equally for both groups. For
using OCT signal. coated groups, the distance to non-significantly dif-
Interestingly, we were able to detect changes in ferent fluorescence was shorter than for uncoated
the silicon probe’s position relative to the OCT probe groups. That is, the maximum distance with fluor-
(visible in all animals in figure S1 and highlighted in escence significantly different from control was
7J. Neural Eng. 18 (2021) 045002 I Dryg et al
Figure 3. Effect of CH4 plasma coating on glial encapsulation of the OCT probes. Shown are quantified encapsulation by (a)
astrocytes labeled by GFAP, and (b) microglia labeled by Iba1, with representative uncoated and coated OCT probe images. The
maximum distance with fluorescence intensity significantly different from control tissue is plotted in (c).
lower for coated implants than uncoated implants intravascular implants (Bergmann 2015). However,
(figure 3(c) and significance notations in figures 3(a) our study revealed no clear differences between
and (b)). CH4 -coated or uncoated probes by OCT Imaging
Table S1 summarizes the comparisons and statist- or by IHC. This may be explained by the differ-
ical tests that were performed in this section, along ences between the intravascular and nervous system
with a post-hoc power analysis showing that the environments, and by the flow component present
uncoated vs coated direct comparisons were lacking intravascularly but not in brain tissue. Also, the afore-
in power, and the uncoated vs control and coated vs mentioned study used titanium as the base material,
control comparisons were sufficiently powered. Given whereas this study used glass. The fluorescence of
that there were only three animals per group, the CH4 coated probes stopped being significantly dif-
sample size should be increased to be able to prop- ferent from controls closer than uncoated probes,
erly test the differences between coated and uncoated suggesting the glial capsule may be smaller around
groups. CH4 coated probes. However, the variance was gen-
In a previous implant study, CH4 plasma coat- erally high for both groups. Further study with larger
ings reduced tissue encapsulation of titanium sample sizes need to be performed to elucidate any
8J. Neural Eng. 18 (2021) 045002 I Dryg et al
relationship between CH4 plasma coatings and the analysis of silicon-based intracortical microelectrode arrays
foreign body reaction in the brain. in non-human primates J. Neural Eng. 10 066014
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Acknowledgments on inflammation and neuronal health at the electrode-tissue
interface in rat spinal cord and dorsal root ganglion Acta
This work is partially supported by the BrainLinks- Biomater. 8 3561–75
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