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COMMUNICATION 1800427 (1 of 6) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advopticalmat.de Ultrasoft and Highly Stretchable Hydrogel Optical Fibers for In Vivo Optogenetic Modulations Lulu Wang, Cheng Zhong, Dingning Ke, Fengming Ye, Jie Tu, Liping Wang,* and Yi Lu* DOI: 10.1002/adom.201800427 and the elucidation of neuropsychiatric disease mechanisms. [1,2] Recent optoge- netics developments have provided a potential treatment of epilepsy, [3,4] Par- kinson’s disease, [5,6] depression, [7,8] and other brain disorders, [9–11] where the cell type-specific modulation of neural circuits may address the pathological symptoms. [12] To manipulate the activi- ties of specific neurons or neural circuits in vivo using optogenetics, implantable optical waveguides are frequently used to deliver laser light into the virus-infected brain regions. [13,14] Primarily, these wave- guides are silica optical fibers, with an average Young’s modulus at least six orders of magnitudes larger than that of the neural tissues. [15–17] The elastic mis- match between the silica optical fibers and organisms may lead to the host tissue injuries, which subsequently induce neu- ronal death in the implant surroundings. To prevent this, stretchable and flexible optoelectronic implants [18–21] and polymer integrated probes [22–25] have been devel- oped, with a decreased tissue response and consistent performance during chronic implantations. However, the complex and expensive fabrication process of the implantable optoelectronics and the relatively low deformability of the polymer integrated probes may hinder their widespread applications. Recent advances in hydrogel-based optical fiber provide a low propagation loss waveguide with much higher stretchability and lower Young’s modulus, [26–28] which may largely eliminate the effects of the mechanical mismatch and tissue movements. [15,29] However, the feasibility and advantages of the hydrogel optical fiber use for chronic optogenetic brain modulation in free-moving ani- mals have not been systematically investigated. Here, we developed a simplified method for the fabrication of low-modulus and high-stretchable alginate-polyacrylamide (PAAm) hydrogel optical fibers and investigated their applica- tion in vivo. The alginate-PAAm precursor was polymerized and cross-linked in a simplified one-step process, while its molecular structure was confirmed using the Raman spectra analysis (Figure 1a). With the increase in the PAAm contents, main band intensity increased as well: 1101 cm 1 at the NH 2 twisting, 1322 cm 1 at the CH 2 wagging, 1429 cm 1 at the CN vibration, 1452 cm 1 at the CH 2 bending, 1621 cm 1 at the CO stretching vibration, and 1677 cm 1 at the NH 2 Optogenetics has been widely applied as a cell-specific technique with high temporal resolution for the modulation of neural circuitry in vivo, offering potential novel treatments for neuropsychiatric diseases. However, to date, the most widely used optogenetics waveguides remain silica optical fibers, which may lead to a mismatch in the mechanical properties between the implants and neural tissues. To resolve this issue, alginate-polyacrylamide hydrogel optical fibers can be fabricated in a simplified one-step process, and they show significantly improved characteristics for the in vivo optogenetic applications, including low light-propagation loss and Young’s modulus, and high stretchability. After the expression of AAV-CaMKIIα-ChR2-mCherry, blue light pulses are delivered into hippocampus using a hydrogel-optrode array, and frequency-dependent neural responses can be observed. Moreover, optogenetic stimulation through the chronic implanted hydrogel optical fibers in the primary motor cortex can considerably modulate the animal’s behavior. Hydrogel fibers significantly alleviate tissue response at the implant/neural interface, compared with that observed using the silica optical fibers. Taken together, the results of this study demonstrate the feasibility and advantages of the hydrogel optical fiber use for chronic optogenetic modulation in free- moving animals. Hydrogel implant use may allow the development of novel therapeutic strategies for the treatment of neuropsychiatric disorders. L. Wang, Dr. C. Zhong, F. Ye, Prof. J. Tu, Prof. L. Wang, Dr. Y. Lu Collaborative Innovation Center for Brain Science CAS Center for Excellence in Brain Science and Intelligence Technology The Brain Cognition and Brain Disease Institute Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences Shenzhen 518055, China E-mail: [email protected]; [email protected] L. Wang, F. Ye Shenzhen College of Advanced Technology University of Chinese Academy of Sciences Shenzhen 518055, China Dr. D. Ke Experiment and Innovation Center Harbin Institute of Technology Shenzhen Graduate School Shenzhen 518055, China The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adom.201800427. Hydrogel Optical Fibers Optogenetics allows a precisely timed control of specific neuron subtypes in free-behaving animals, which has been widely applied for the determination of neural circuit characteristics Adv. Optical Mater. 2018, 1800427
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Page 1: Ultrasoft and Highly Stretchable Hydrogel Optical Fibers ...wanglab.siat.ac.cn/wanglab_en/upload/publications/... · of low-modulus and high-stretchable alginate-polyacrylamide (PAAm)

COMMUNICATION

1800427 (1 of 6) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advopticalmat.de

Ultrasoft and Highly Stretchable Hydrogel Optical Fibers for In Vivo Optogenetic Modulations

Lulu Wang, Cheng Zhong, Dingning Ke, Fengming Ye, Jie Tu, Liping Wang,* and Yi Lu*

DOI: 10.1002/adom.201800427

and the elucidation of neuropsychiatric disease mechanisms.[1,2] Recent optoge-netics developments have provided a potential treatment of epilepsy,[3,4] Par-kinson’s disease,[5,6] depression,[7,8] and other brain disorders,[9–11] where the cell type-specific modulation of neural circuits may address the pathological symptoms.[12] To manipulate the activi-ties of specific neurons or neural circuits in vivo using optogenetics, implantable optical waveguides are frequently used to deliver laser light into the virus-infected brain regions.[13,14] Primarily, these wave-guides are silica optical fibers, with an average Young’s modulus at least six orders of magnitudes larger than that of the neural tissues.[15–17] The elastic mis-match between the silica optical fibers and organisms may lead to the host tissue injuries, which subsequently induce neu-ronal death in the implant surroundings. To prevent this, stretchable and flexible optoelectronic implants[18–21] and polymer integrated probes[22–25] have been devel-oped, with a decreased tissue response and consistent performance during

chronic implantations. However, the complex and expensive fabrication process of the implantable optoelectronics and the relatively low deformability of the polymer integrated probes may hinder their widespread applications. Recent advances in hydrogel-based optical fiber provide a low propagation loss waveguide with much higher stretchability and lower Young’s modulus,[26–28] which may largely eliminate the effects of the mechanical mismatch and tissue movements.[15,29] However, the feasibility and advantages of the hydrogel optical fiber use for chronic optogenetic brain modulation in free-moving ani-mals have not been systematically investigated.

Here, we developed a simplified method for the fabrication of low-modulus and high-stretchable alginate-polyacrylamide (PAAm) hydrogel optical fibers and investigated their applica-tion in vivo. The alginate-PAAm precursor was polymerized and cross-linked in a simplified one-step process, while its molecular structure was confirmed using the Raman spectra analysis (Figure 1a). With the increase in the PAAm contents, main band intensity increased as well: 1101 cm−1 at the NH2 twisting, 1322 cm−1 at the CH2 wagging, 1429 cm−1 at the CN vibration, 1452 cm−1 at the CH2 bending, 1621 cm−1 at the CO stretching vibration, and 1677 cm−1 at the NH2

Optogenetics has been widely applied as a cell-specific technique with high temporal resolution for the modulation of neural circuitry in vivo, offering potential novel treatments for neuropsychiatric diseases. However, to date, the most widely used optogenetics waveguides remain silica optical fibers, which may lead to a mismatch in the mechanical properties between the implants and neural tissues. To resolve this issue, alginate-polyacrylamide hydrogel optical fibers can be fabricated in a simplified one-step process, and they show significantly improved characteristics for the in vivo optogenetic applications, including low light-propagation loss and Young’s modulus, and high stretchability. After the expression of AAV-CaMKIIα-ChR2-mCherry, blue light pulses are delivered into hippocampus using a hydrogel-optrode array, and frequency-dependent neural responses can be observed. Moreover, optogenetic stimulation through the chronic implanted hydrogel optical fibers in the primary motor cortex can considerably modulate the animal’s behavior. Hydrogel fibers significantly alleviate tissue response at the implant/neural interface, compared with that observed using the silica optical fibers. Taken together, the results of this study demonstrate the feasibility and advantages of the hydrogel optical fiber use for chronic optogenetic modulation in free-moving animals. Hydrogel implant use may allow the development of novel therapeutic strategies for the treatment of neuropsychiatric disorders.

L. Wang, Dr. C. Zhong, F. Ye, Prof. J. Tu, Prof. L. Wang, Dr. Y. LuCollaborative Innovation Center for Brain ScienceCAS Center for Excellence in Brain Science and Intelligence TechnologyThe Brain Cognition and Brain Disease InstituteShenzhen Institutes of Advanced TechnologyChinese Academy of SciencesShenzhen 518055, ChinaE-mail: [email protected]; [email protected]. Wang, F. YeShenzhen College of Advanced TechnologyUniversity of Chinese Academy of SciencesShenzhen 518055, ChinaDr. D. KeExperiment and Innovation CenterHarbin Institute of Technology Shenzhen Graduate SchoolShenzhen 518055, China

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adom.201800427.

Hydrogel Optical Fibers

Optogenetics allows a precisely timed control of specific neuron subtypes in free-behaving animals, which has been widely applied for the determination of neural circuit characteristics

Adv. Optical Mater. 2018, 1800427

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bending.[30,31] Swelling of the hydrogel is crucial for its in vivo applications, as it affects the inert physical characteristics and insertion trauma during implantation. Hydrogel samples were dehydrated and immersed into artificial cerebrospinal fluid (ACSF) for 72 h, and their weights were determined. The average linear expansion ratios of the fully swollen alginate-PAAm hydrogels were 1.900–1.927, comparable to those in the fully dehydrated states (Figure 1b) and almost independent of the AAm concentration. The refractive index of the fully swollen hydrogel slightly increased from 1.3454 with the AAm content and reached the platform at ≈1.3533 (Figure 1c).

We fabricated hydrogel optical fibers with various diameters using different tube molds (Figure S1, Supporting Informa-tion). To balance the size and performance, including both mechanical and optical characteristics, a hydrogel optical fiber obtained by polymerizing the alginate-PAAm precursor in a silicone rubber tube (inner diameter = 300 µm) was used. Hydrogel fiber was dehydrated, threaded through an optical ceramic ferrule (inner diameter = 300 µm), and immersed in ACSF for 2 h before use. The fully swollen hydrogel fiber holds the stiff ceramic ferrule steadily, forming a smooth optical connection (Figure 1d). Blue laser light (λ = 472 nm), fre-quently used for the excitation of channelrhodopsin-2 (ChR2)-expressing neurons, was conducted into the hydrogel fibers through silica optical fibers (diameter = 200 µm, NA = 0.37) terminated with a ceramic connector (Figure 1e). The power density of the light transmitted through the hydrogel optical fibers fabricated using different AAm concentrations was determined (Figure S2, Supporting Information), and their propagation loss (dB cm−1) was calculated (Figure 1f). The result indicates that the propagation loss of the fully swollen hydrogel optical fibers somewhat decreased with an increase in the PAAm content, reaching a minimum value (0.249 dB cm−1) in hydrogel fibers fabricated with 40 wt% of the AAm in pre-cursor solution. However, further increase in PAAm led to a

sharp increase in the propagation loss, probably due to a more compact polymer network of semicross-linked hydrogels. Addi-tionally, the mechanical properties of the fully swollen hydrogel fibers were characterized by Young’s modulus, which slowly increased from 48.234 to 90.849 kPa with the AAm concentra-tion increase (Figure 1g), showing that the Young’s modulus of the hydrogel fibers is significantly lower than that of the con-ventional silica optical fibers (≈10 GPa), and more compatible with the neural tissues (≈1 kPa).[15–17,29] Therefore, due to its swelling ratio and Young’s modulus, as well as low propagation loss, hydrogel optical fibers fabricated with 40% AAm concen-tration were selected for further studies.

The feasibility of the hydrogel optical fiber use for in vivo optogenetic modulations was examined. The swollen hydrogel optical fiber showed excellent elastic stretchability, and its con-ductivity dropped only 13.97 and 30.15% when stretched to 120 and 140% of its initial length, respectively (Figure S3, Sup-porting Information), sufficient to meet brain or neural tissue deformation. Furthermore, the fabricated hydrogel fiber exhib-ited high optical conductivity even in the aqueous medium, suit-able for in vivo guiding of the laser light (Figure S4, Supporting Information). To determine whether the fabricated hydrogel optical fibers deliver enough light to excite glutamatergic neu-rons, adeno-associated virus (AAV)-CaMKIIα-ChR2-mCherry was injected into C57 mouse hippocampus (Figure 2a). A custom-made hydrogel-optrode array containing four stere-otrodes was used for optogenetic stimulation and electrical recording in vivo (Figure 2b; and Figure S5, Supporting Infor-mation). The hippocampal neurons were transduced with AAV 4 weeks after the injection, and blue light pulses (20 Hz, 5 ms) were delivered for optogenetic activation. As the implantation depth was only 2.0 mm, therefore the diameter-to-length ratio of the hydrogel optical fiber was relatively high. This is benefi-cial for decreasing propagation loss during optical stimulation. Representative examples of raw spike data showed that each

Adv. Optical Mater. 2018, 1800427

Figure 1. Chemical and physical properties of alginate-PAAm hydrogels. a) Raman spectra of prepared hydrogels. b) Linear expansion ratios of hydrogel samples after swelling, determined from the cube root of the swelling ratio (n = 6). c) Refractive index of swollen hydrogels (n = 6). d,e) Representa-tive images of a dehydrated (d) and swollen (e) hydrogel optical fiber coupled with an optical ceramic ferrule. f) Blue light (λ = 472 nm) propagation loss of the swollen hydrogel optical fibers (n = 12). g) Young’s modulus of swollen hydrogel optical fibers (n = 10). In (b), (c), (f), and (g), data are presented as mean ± standard error of the mean.

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flash of the pulse train generated a large electrophysiological response by the activated neurons (Figure 2c). 3D views of the unit clusters and averaged spike waveforms of the recorded neurons were also analyzed. Two types of neurons activated by hydrogel optical fiber were clearly observed (Figure 2d), and the peak-to-peak amplitudes of these sorted waveforms were 150–200 µV (Figure 2e), implying that the fabricated hydrogel optical fibers can be used as waveguides for optogenetic stimu-lation in vivo.

We further investigated the long-term performance of hydrogel optical fibers in vivo, by implanting them into the pri-mary motor cortex (M1) of mice after injecting AAV-CaMKIIα-ChR2-mCherry (Figure 2f; and Figures S6 and S7, Supporting Information). The mice could freely explore an open-field arena for 10 min without optical stimulation (Figure 2g; and Video S1, Supporting Information). Afterward, we examined whether the blue light pulses (10 mW, 20 Hz, 5 ms duration) in the implanted hydrogel optical fibers can activate the M1 glutamatergic neurons and subsequently modulate animal behavior. These mice exhibited increased right-turning and rotating movements immediately after light delivery (Figure 2h; and Video S2, Supporting Information), attributable to the con-tralateral M1 neuron activation. A significant decrease in the total moving distance during optogenetic modulation period was observed (Figure 2i), in accordance with the behavioral results obtained on mice implanted with conventional silica optical fibers (Figure S8, Supporting Information).

Furthermore, to examine the long-term functional sta-bility and tissue-compatibility of the hydrogel optical fibers, samples were implanted into the deep brain of C57 mice. We found that the stretchability and optical conductivity of the hydrogel fibers were not significantly affected 4 weeks after implantation (Figure S9, Supporting Information). The tissue response to the implants was characterized by glial fibril-lary acidic protein (GFAP) immunoreactivity (Figure 3a),

and reactivated astrocytes occupied the zone around the silica fiber implant, while a much lighter GFAP-positive zone was shown to represent hydrogel surroundings. Quantitative anal-ysis of GFAP intensity at the silica and hydrogel optical implants as a function of distance from the interface is presented in Figure 3b, indicating that the GFAP intensity in the hydrogel group was significantly lower (p < 0.005) than that of the silica group, along 175 µm to the implant interface. Neuronal sur-vival around the implants was assessed by analyzing neuronal nucleus (NeuN) immunoreactivity. Severe neuronal loss was observed adjacent to the conventional silica optical fiber, but not at the hydrogel/tissue interface (Figure 3c). Quantitative analysis demonstrated that the neuronal density in the hydrogel optical fiber group remained almost unchanged across 500 µm distance 4 weeks after implantation, significantly higher (p < 0.005) than that observed in the silica optical fiber group in the test zone within 100 µm from the implant/tissue interface (Figure 3d). This suggests that the hydrogel optical fibers are suitable for chronic optogenetic modulations in vivo, with a decreased glial encapsulation and an improved neuronal viability around the implants. However, it is worth mentioning that the size of the fabricated hydrogel fibers should be further decreased at this stage to meet the requirements of multisite optical stimula-tion and minimal implantation trauma. Some issues, such as novel core and cladding materials, and improved optical implant designs, still require further systematic investigation.

In conclusion, we demonstrated the feasibility and advan-tages of an alginate-PAAm hydrogel optical fiber use for chronic optogenetic brain modulation in free-moving animals. This fiber was polymerized in a one-step process, exhibiting a low-modulus and high-stretchable properties compatible with bio-logical tissues. Optical ceramic ferrule-coupled hydrogel fiber implants were fabricated by a simplified swollen-fixing strategy, which facilitates their combination with electrode arrays and in vivo optogenetic stimulation applications. Owing to the low

Adv. Optical Mater. 2018, 1800427

Figure 2. Optogenetic modulations in vivo. a) Expression of CaMKIIα-ChR2-mCherry (red) in hippocampal neurons 4 weeks after injection (bar = 500 µm). b) A schematic diagram of a hydrogel optical fiber-coupled electrode (hydrogel-optrode) array. c) Electrophysiological recordings before and during optogenetic stimulation (blue bar) using hydrogel-optrode array in vivo. d) Principal-component analysis of two separable neuronal units pre-sented in (c). The points are colored according to the assigned cluster, and low-amplitude spikes are not shown. e) Waveforms of the recorded units presented in (c). f) Expression of CaMKIIα-ChR2-mCherry (red) in the M1 4 weeks after injection (bar = 200 µm). g,h) Representative moving traces in an open field before (g) and during (h) optogenetic modulation. i) Total distance travelled in the open field (n = 6, ***p < 0.005, t-test).

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blue-light propagation loss of the fully swollen hydrogel fiber, we observed light-evoked and frequency-dependent responses of the hippocampal neurons using a hydrogel-optrode array. Furthermore, we demonstrated that light delivery through the chronic implanted hydrogel optical fibers can activate the AAV-expressing neurons in M1, modulating animal behavior. Hydrogel optical fibers significantly alleviated tissue response and improved neuronal survival at the implant/tissue inter-face, in contrast to those of the conventional silica optical fibers. Although we presented only some of the advantages of hydrogel optical fibers, these low-modulus, highly stretchable, and biocompatible implants have shown great potential for the therapeutic optical stimulation of neural tissue with large defor-mations, including the spinal cord, sciatic, and vagus nerve. Our approach may help expand the potential applications of optogenetics and provide novel insights into the future thera-peutic treatments of neuropsychiatric disorders.

Experimental SectionHydrogel Optical Fiber Fabrication: Hydrogel optical fiber precursors

were synthesized by mixing the aqueous solutions of AAm (20, 30, 40, and 50 wt%) with 2 wt% sodium alginate, N,N-methylenebisacrylamide (0.15 wt% of AAm weight), ammonium persulfate (APS, 0.4 wt% of AAm weight), and N,N,N′,N′-tetramethylethylenediamine (TEMED, 1000 ppm; all, Sigma-Aldrich). The precursor was cooled in an ice-bath, degassed, and then injected into a tube mold (inner diameter = 75, 150, or 300 µm). The filled silicone tube was transferred into a glass container and cross-linked at 60 °C in the absence of oxygen for 30 min. Following this, the hydrogel fiber was dehydrated and extracted from the tube. To facilitate experimental measurements, the dehydrated hydrogel fiber was threaded through an optical ceramic ferrule and allowed to swell in water. Alginate-PAAm hydrogel bulk samples (10 × 10 mm,

5 mm thick) were prepared for the Raman, swelling ratio, and refractive index analyses.

Chemical and Physical Characterizations of Hydrogels: The chemical composition of the bulk hydrogels was characterized using Raman microspectrometer (Renishaw RM1000, UK) with a spectral resolution of 1 cm−1 in the 1800–400 cm−1 range. Dry hydrogel samples were immersed into ACSF for 72 h to reach swelling equilibrium, and the weight and refractive index were measured. The swelling ratio (SR) is defined as SR = (Ws − Wd)/Wd, where Ws and Wd are the weight of swollen and dry hydrogel samples, respectively. The Young’s modulus (E) of swollen hydrogel optical fibers was calculated as E = (F/A)/(ΔL/L0), where F is the force exerted on hydrogel fiber, A is the cross-sectional area, ΔL is the change of the length, and L0 is the original length of hydrogel fiber. Light propagation loss was measured using the cutback technique.[16] Blue laser light (λ = 472 nm) was conducted into the swollen hydrogel fiber, and the power density of light transmitted through the hydrogel optical fiber was gauged. The measurement was repeated each time after removing the end of the hydrogel fiber (ΔL = 2 cm). The propagation loss (PL; dB cm−1) was calculated as PL = −10[Δlog(Power)]/ΔL.

Optical Stimulation and Electrophysiological Recordings In Vivo: All experiments were performed in accordance with the protocols approved by the Ethics Committee for Animal Research, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Wild-type male, 8-week-old C57 mice were used in the studies. A hole was carefully drilled at anteroposterior −2.06 mm, mediolateral −1.35 mm, and AAV9-CaMKIIα-ChR2-mCherry was injected into hippocampus at dorsoventral (Hesdorffer, #19) −2.0 mm. Eight-channel microwire electrode arrays were generated as previously described.[3,14] A hydrogel optical fiber was coupled with the electrode to form a hydrogel-optrode array (Figure S5, Supporting Information). Four weeks after the AAV injection, a hydrogel-optrode array was lowered into the hippocampus (DV −2.0 mm), and 472 nm laser light pluses (5 ms at 20 Hz) were used for ChR2 activation. Electrophysiological recordings were performed using a multichannel neural acquisition processor (Plexon). Data in all recording channels were sampled at 40 kHz and bandpass filtered at 300–5000 Hz. To verify the expression of AAV, 35 µm thick coronal cortical slices were obtained

Adv. Optical Mater. 2018, 1800427

Figure 3. Inflammatory response and neuronal survival around optical implants. a,c) GFAP (a) and NeuN (c) immunostaining of conventional silica and hydrogel optical fibers (red: GFAP; green: NeuN; blue: DAPI; bar = 200 µm) at 4 weeks after implantation. b,d) Quantitative comparisons of GFAP (b) and NeuN (d) immunoreactivity between conventional silica and hydrogel optical fibers; comparisons were performed using intensity profiles as a function of distance from the implant interface, shown as mean values ± standard error of the mean (GFAP, n = 16; NeuN, n = 12). Shaded area, significant difference (p < 0.005, t-test).

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using a cryostat microtome (Leica CM1950), and ChR2-mCherry expressing hippocampal neurons were observed under an Olympus fluorescence microscope (VS120).

Optogenetic Modulation of Mouse Behavior: To investigate the long-term performance of hydrogel optical fiber implants, AAV-CaMKIIα-ChR2-mCherry was injected into the M1 of C57 mice at anteroposterior +1.98 mm, mediolateral −1.80 mm, and DV −1.5 mm. Afterward, a hydrogel optical fiber was implanted into the M1 (DV −1.5 mm), and fixed to the skull with dental cement. The end of each optical ceramic ferrule was sealed with a plastic cap to keep the hydrogel fiber moist prior to use (Figure S6, Supporting Information). During the testing procedure, the mice were allowed to freely move in an open field chamber (60 × 60 × 50 cm) for 10 min and then 472 nm light pulses (10 mW, 20 Hz, 5 ms duration) were delivered into the M1 through the implanted hydrogel optical fiber for additional 10 min. Mouse behavior was recorded with digital video camera and analyzed by Any-maze video tracking system.

Chronic Tissue Response Assessment: Hydrogel fibers and conventional silica optical fibers were implanted into the M1 of C57 mice, respectively. At 4 weeks after implantation, the mice were sacrificed and horizontal sections (35 µm thick) were prepared. Antibodies against GFAP (Abcam) and NeuN (Abcam) were used to label astrocytes and mature neurons, respectively. Fluorescence images were obtained using an Olympus VS120 microscope. Quantitative analyses were performed using a custom software developed in MATLAB (MathWorks, USA).[32] The staining intensity of GFAP and NeuN were calculated as a function of distance to the implant surface. The results shown represent the average intensity profiles of the analyzed area within 500 µm from the implant/tissue interface.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsL.W. and C.Z. contributed equally to this work. This research was partially sponsored by the National Natural Science Foundation of China (Nos. 81425010, 31630031, and 31700921), the Strategic Priority Research Program of the CAS (No. XDB02050003), the CAS-SAFEA International Partnership Program for Creative Research Teams (No. 172644KYS820170004, 172644KYSB20160057), the External Cooperation Program of the CAS (No. GJHZ1508), the Youth Innovation Promotion Association of the CAS, the Shenzhen Governmental Research Grants (Nos. JSGG20160429184327274, LSGG20160428140402911, JCYJ20160429190927063, JCYJ20150529143500959, and JCYJ20150401150223647), the Shenzhen Engineering Lab of Brain Activity Mapping Technologies, the Shenzhen Discipline Construction Project for Neurobiology, and the Guangdong Key Lab of Brain Connectome.

Conflict of InterestThe authors declare no conflict of interest.

Keywordshydrogels, neural modulation, optical fibers, optogenetics, stretchable

Received: March 31, 2018Revised: May 5, 2018

Published online:

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