Fabrication and characterizations of POFs
To facilitate light transmission in the biological environment, the POF was composed of a core/clad structure. PDMS fibrous waveguides were constructed as the fiber cores via a thermal drawing process (Fig. 1A). As PDMS can be quickly cured (~ 2 s) at 280 °C, fiber cores with a wide range of feature dimensions (diameter = 100–500 μm) can be created at different pulling-up speeds of the pre-polymer (Fig. 1B). To balance the implantation trauma and optical performance of the POFs, fiber cores with a diameter of 200 μm were used in this study. Besides, poly(vinyl alcohol)/poly(acrylic acid) interpenetrating polymer network (PVA/PAA IPN) with a low RI of 1.3440 and a linear swelling ratio of 2.75 was synthesized as a cladding material (Additional file 1: Fig. S1 and S2A). Then, an oxygen plasma-treated PDMS fiber core was coated with the prepared PVA/PAA hydrogel solution, dehydrated, and re-swollen in an artificial cerebrospinal fluid (ACSF). Subsequently, a homogeneous hydrogel cladding layer was formed on the surface of the PDMS optical fiber core (Additional file 2: Fig. S2B).
To form a smooth optical connection for in vivo optogenetics, the fabricated POF was coupled with an optical ceramic ferrule and immersed in ACSF prior to use. The elastic POF showed a linear stress-strain relationship up to 150% strain and a Young’s modulus of 1.22 MPa (Additional file 3: Fig. S3A), which can steadily hold the stiff ceramic ferrule under 100% stretching deformation (Fig. 1C, Additional file 13: Movie 1). Four hundred seventy-two nanometers of blue laser light, commonly used for channelrhodopsin-2 (ChR2) excitation, was transmitted into the POF through a commercial SOF (diameter = 200 μm, NA = 0.37) terminated with a ceramic connector (Fig. 1D). To investigate the mechanical stability of those fabricated waveguides, we test the output power of POFs before and after repeated stretching to 200% of their original length for 10,000 times and found no significant influence on their optical transmission capability (Additional file 4: Fig. S4A). As the average RIs of the PDMS core (ncore) and PVA/PAA cladding (nclad) were 1.4109 and 1.3440 respectively (n = 6, Fig. 1E), the theoretical numerical aperture (NA = [ncore2 − nclad2]0.5) of the fabricated POF was 0.4293. In addition, the blue-light propagation loss of the POFs measured in air (nair = 1.000) using a cutback technique [33, 35] was 1.018 dB·cm−1 (Additional file 5: Fig. S5). On comparison with the light powers obtained in air, it was observed that our POFs can retain approximately 90% of the optical transmission capacity (Additional file 6: Fig. S6) in water (nwater = 1.333, similar to the RI of brain tissues), which can meet the requirements of different application scenarios.
Long-term biocompatibility and stability of POFs
To examine the long-term biocompatibility of the POFs, samples were implanted into the brain of C57 mice for 4 weeks (Fig. 2A). Reactivated astrocytes occupied the zone around the SOF implants, whereas a considerably lighter glial fibrillary acidic protein (GFAP) positive zone was adjacent to the interface of the POFs. A quantitative analysis of the GFAP intensity as a function of the distance from the interface is presented in Fig. 2B, which shows that the GFAP intensity in the POF group was significantly lower than that of the SOF group (n = 8 mice, p < 0.005, t test), along 220 μm to the implant interface. Furthermore, the neuronal survival around the implants was assessed by analyzing the neuronal nucleus (NeuN) immunoreactivity. The neuronal density for the POF group was significantly higher than that of the SOF group in the test zone (n = 8 mice, p < 0.005, t test), within 40 μm of the implant-tissue interface (Fig. 2C). We also investigated the long-term physical stability of those fabricated waveguides. After the 4 weeks implantation, the Young’s modulus of the POF was 1.19 MPa, similar to the value before implantation (Additional file 3: Fig. S3B). Besides, no significant influence on the optical transmission capability of the POFs was observed after the long-term implantation (Additional file 4: Fig. S4B). These results suggest the long-term stability and validity of the fabricated POFs.
Chronic optogenetic verifications
To determine whether the POFs could deliver enough light for optogenetic modulations in vivo, adeno-associated virus (AAV)-CaMKIIα-ChR2-mCherry was injected into the hippocampus of a C57 mouse (Additional file 7: Fig. S7). After the viral expression, a custom-made optrode array containing a POF and two tetrodes was used for in vivo optogenetic stimulation and electrophysiological recording. Continuous, frequency-dependent action potentials were detected when light pulses (472 nm, 20 Hz, 5 ms duration) were delivered to the hippocampus, suggesting that the fabricated POFs can serve as waveguides for in vivo optogenetic stimulation. To further investigate the long-term validity of these optical waveguides, POFs were implanted into the primary motor cortex (M1) 1 week after AAV-CaMKIIα-ChR2-mCherry injection (Fig. 3A). Four weeks after viral injection, blue light pulses (20 Hz, 5 ms duration) were conducted through the implanted POFs to activate the ChR2-expressing neurons in the M1, and continuous action potentials of the optically activated neurons were recorded (Fig. 3B, C). Interestingly, we also observed that the optogenetic stimulation caused motor coordination impairment in the mice (Fig. 3D), including increased turning, rotating, and falling behaviors. Consequently, the total moving distance during M1 activation (15.15 ± 3.96 m) was significantly decreased (n = 7, p < 0.005, t test) compared to the behavioral results obtained on the mice before optogenetic stimulation (21.46 ± 3.83 m) (Fig. 3E).
Effect of VNOS on physiological responses
To study the feasibility of using our fabricated waveguide for chronic VNOS in free-moving rodents, we implanted the POF under the mouse skin, and fixed the ceramic ferrule connector to the skull with dental cement for in vivo optogenetic stimulation (Fig. 4A). The tip of the POF was placed towards to the left vagal ganglion of VGAT-ChR2 transgenic mouse and fixed in place with 3M tissue glue to construct a smooth and stable optical connection (Additional file 8: Fig. S8). A micro-wire stereotrode was implanted in the vagal ganglion to record the electrophysiological responses during light delivery (472 nm, 130 Hz, 5 ms duration). We found that VNOS selectively activated the fast-spiking γ-aminobutyric acid (GABA)-ergic interneurons, while suppressed the broad-spiking cholinergic neurons (Fig. 4B–D).
Furthermore, the heart rates (HRs) of the mice were monitored to investigate the influence of VNOS on innate physiological responses. It is worth mentioning that, as the frequency for the VNOS exhibits a dose-dependent inhibitory effect on the cardiac activity of the VGAT-ChR2 transgenic mouse, 130 Hz was chosen as an optimized stimulation frequency for the modulation of the vagus nerve in this study (Additional file 9: Fig. S9). Under anesthesia, the average HR of the mice was 555.5 ± 62.7 beats per minute (bpm), which dramatically decreased (n = 5, p < 0.005, t test) during optical stimulation and reached a plateau of 322.7 ± 76.4 bpm (Fig. 4E, F). To investigate the validity and reliability of the POFs for optogenetic stimulation under spontaneous tissue deformations, we monitored the HR in freely behaving animals before and during VNOS. The HRs recorded in awaken mice were higher than those obtained in anesthetized mice, with an average value of 694.4 ± 42.3 bpm. During light delivery, the average HR of the experimental subjects was significantly decreased (n = 8, p < 0.005, t test) to 643.8 ± 62.0 bpm (Fig. 4G, H).
Effect of VNOS on behaviors
To study the influence of VNOS on animal behaviors, we performed an open field test after POF implantation (Fig. 5A). We observed that the total distance traveled in the open field arena (OFA) reduced (n = 8, p < 0.01, t test) from 26.03 ± 4.96 m to 19.53 ± 7.73 m during optogenetic stimulation, while the total entries to and time spent in the center zone of the OFA were not significantly changed (n = 8, p > 0.05, t test) (Fig. 5B). To further investigate the effect of VNOS on emotion-related behaviors in mice, we then performed elevated plus-maze (EPM) test (Fig. 5C). Although optogenetic stimulation showed no significant influence (n = 8, p > 0.05, t test) on the open arm entries of the experimental subjects, the time spent on the open arms increased (n = 8, p < 0.01, t test) from 29.48 ± 13.71 s to 47.58 ± 11.87 s (Fig. 5D). In addition, we also monitored the HRs of experimental subjects during EPM test. We observed that the HRs increased (n = 4, p < 0.05, t test) from 677.59 ± 34.29 bpm to 711.29 ± 47.45 bpm when the mice moved from the closed arm to the open arm, which instantly dropped back to normal level (668.83 ± 62.87, n = 4) under vagal modulations (Additional file 10: Fig. S10).
Next, we adopted an unpredictable chronic mild stress (UCMS) model to further investigate the effect of VNOS on animals’ behaviors. After the UCMS treatment, the total entries to the center zone of the OFA decreased (n = 4, p < 0.05, t test) from 27.00 ± 7.16 times to 10.00 ± 2.94 times, and the time spent in the center zone decreased (n = 4, p < 0.05, t test) from 69.45 ± 27.20 s to 21.05 ± 5.65 s (Additional file 11: Fig. S11A, B). Besides, the open arm entries of the animals decreased (n = 4, p < 0.05, t test) from 11.00 ± 6.98 times to 4.00 ± 2.94 times, and the time spent on the open arms also decreased (n = 4, p < 0.01, t test) from 41.38 ± 15.19 s to 7.15 ± 5.70 s (Additional file 11: Fig. S11C, D). Then, we investigated the effect of VNOS on the anxiety-like behaviors of the experimental subjects. Interestingly, during VNOS, the total entries to the center zone of the UCMS-treated animals increased (n = 8, p < 0.005, t test) from 10.38 ± 2.83 times to 13.75 ± 2.87 times, and the time spent in the center zone increased (n = 8, p < 0.005, t test) from 21.03 ± 8.31 s to 50.30 ± 15.11 s (Fig. 6A, B). Besides, the open arm entries of the animals increased (n = 8, p < 0.005, t test) from 4.50 ± 2.51 times to 12.25 ± 6.67 times, and the time spent on the open arms increased (n = 8, p < 0.005, t test) from 9.38 ± 6.99 s to 38.83 ± 18.68 s (Additional file 11: Fig. S11C, D).
Furthermore, to study the long-term influence of VNOS, behaviors of the UCMS-treated animals were monitored each week. After repeated VNOS treatments, we found that the animals exhibited an increased tendency to explore the center zone of the OFA and the open arms of the EPM, respectively (Additional file 11: Fig. S11). In addition, after long-term VNOS, tissue response around the POFs was studied using hematoxylin-eosin (HE) staining, and no significant cell loss (n = 30 slices from 5 mice, p > 0.05, t test) was observed in the POF-contacted tissues (Additional file 12: Fig. S12). It proves the mechanical compatibility between the stretchable POFs and the soft tissues and implies that the POFs can meet the requirement of flexible optogenetic applications in vivo.