Tannic acid as building block constructing injectable hydrogel and regulating microglial phenotype to enhance neuroplasticity for post-stroke rehabilitation

doi:10.21203/rs.3.rs-3259861/v1

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Research Article

Tannic acid as building block constructing injectable hydrogel and regulating microglial phenotype to enhance neuroplasticity for post-stroke rehabilitation

https://doi.org/10.21203/rs.3.rs-3259861/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 31 Oct, 2023

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Background

Stroke is the second leading cause of mortality and disability in the world. Post-stroke rehabilitation is still unsatisfactory in clinics, which brings giant pains and economic burdens to stroke patients. In this study, an injectable hydrogel where tannic acid (TA) acts as not only a building block but a therapeutic drug was developed for post-stroke rehabilitation.

Methods

TA is used as a building block to form an injectable hydrogel (TA-gel) with carboxymethyl chitosan (CMCS) by multivalent hydrogen bonds. The morphology, rheological property, and TA release behavior of the hydrogel were characterized. The abilities of the TA-gel to modulate microglia (BV2 cells) polarization and subsequently enhance the neuroplasticity of neuro cells (N2A cells) were assessed in vitro. The TA-gel was injected into the cavity of the stroke mouse for the evaluation of motor function recovery, microglial polarization, and neuroplasticity in vivo. The investigation of the molecular pathway through which TA modulates microglia polarization was also explored both in vitro and in vivo.

Results

The TA-gel exhibited a sustainable released behavior of TA. It can suppress the expression of CD16 and IL-1β, and upgrade the expression of CD206 and TGF-β in the oxygen and glucose-deprived (OGD) BV2 cell, indicating the regulation of OGD BV2 cells to anti-inflammatory phenotype in vitro. It further shows the decrease of synaptophysin and PSD95 in the OGD N2a cells is effectively recovered by the anti-inflammatory BV2 cells. Furthermore, it showed the TA-gel can decrease the CD16/iNOS expression, and increase the CD206 expression in the peri-infarct area of stroke mice, implying anti-inflammatory polarization of microglia in vivo. The colocalization of PSD95 and Vglut-1 stains as well as the Golgi stain showed the enhancement of neuroplasticity by the TA-gel. Spontaneously, the TA-gel successfully recovered the motor function of stroke mice. The western blot results in vitro and in vivo suggested TA-gel regulated microglia polarization via the NF-κB pathway.

Conclusion

The TA gel serves as an effective brain injectable implant to treat stroke and shows promising potential to promote post-stroke rehabilitation in the clinic.

Tannic acid

Stroke

Injectable hydrogel

Microglia

Anti-inflammatory phenotype

Stroke is the second leading cause of mortality and disability in the world, following heart disease. ^{[1, 2]} However, post-stroke rehabilitation is still unsatisfactory in clinics, which brings giant pains and economic burdens to stroke patients.^[3] Thus, it is eagerly desirable to develop a novel medical treatment for stroke rehabilitation outside the confines of conventional therapies. Recently, many strategies such as angiogenesis,^[4–6] neurogenesis,^[7–9] neuroplasticity^[10] were carried on to promote post-stroke rehabilitation and have achieved many significant progresses. Microglia, the resident immune cell in the central nervous system plays a significant role in post-stroke rehabilitation since it exerts important effects on spontaneous recovery after stroke, including structural and functional reestablishment of neurovascular networks, neuroplasticity, neurogenesis, axonal remodeling, and blood vessel regeneration.^[11]Our group has focused on the modulation of the microglial polarization^[12–15] in the subacute/chronic phase after stroke and first proposed a novel feasible strategy for post-stroke rehabilitation that enhances the neuroplasticity by regulating the microglial polarization towards anti-inflammatory phenotype.^[16]

As a natural polyphenol, tannic acid (TA) can be extracted from a variety of plants.^[17]It has aroused more and more attention because it is not only a versatile building block to construct multifunction hydrogel with adhesive, stretchability, self-healing properties, and biodegradable ability,^{[18, 19]} but of valuable pharmaceutic effects including anti-inflammatory, antioxidative, and antibacterial.^[20] Jafari, et. al. used TA to post-treat the enzymatically crosslinked chitosan-alginate hydrogels and found the hydrogel adhesiveness, antioxidant, antibacterial properties, as well as hydrogel cytocompatibility and cell proliferation were significantly improved after TA treatment and more suitable for biomedical application.^[21] Guan and Yao prepared a hydrogel with excellent injectable, self-healing, and adhesive properties by facile mixing of quaternary ammonium chitosan (QCS)/TA under physiological conditions.^[22] Benefiting from the inherent antioxidative, antibacterial, and hemostatic abilities of TA and QCS, this hydrogel showed superior reactive oxygen species scavenging activity, broad-spectrum antibacterial ability, and rapid hemostatic capability that cannot only rapidly stop the bleeding of arterial and deep incompressible wounds in mouse tail amputation, femoral artery hemorrhage, and liver incision models but also significantly accelerate wound healing in a full-thickness skin wound model. These results indicate comprehensive utilization of TA’s pharmaceutics effects and its advantages in hydrogel preparation is able to fabricate promising biomedical materials with satisfactory properties.

Considering TA’s outstanding anti-inflammation properties as mentioned above ^{[21, 22]}, we proposed that it might be able to modulate microglia to express anti-inflammatory phenotype after stroke and has the potential to treat stroke and promote post-stroke rehabilitation. However, there are some restrictions in the drug delivery for stroke.^[23] As the gradual recovery of the blood-brain barrier (BBB) in the subacute/chronic phase, the drug with intravenous injection can hardly pass BBB and has a very low bioavailability in the brain.^[24] Although the intracranial injection can directly deliver the drug into the brain, frequent cerebrospinal fluid circulation can soon remove the delivered drug from the brain ^{[25, 26]}. Hence, the treatment of the stroke in the subacute/chronic phase requires a drug carrier that not only can directly deliver the drugs to the brain, but maintain the sustainable release on-site. On the other hand, stroke leads to tissue damage and the formation of an irregular cavity,^{[4, 27]} which represents a potential location for drug delivery.^[28–30] Since the cavity is irregular, an injectable hydrogel that is delivered in liquid form, freely fills the irregular cavity, forms hydrogel in situ, and sustainably releases the drug on-site via diffusion effect is the most suitable delivery system for the stroke in the subacute/chronic phase.^[23]

In this study, TA solution was mixed with carboxymethyl chitosan (CMCS) to obtain an injectable hydrogel (TA gel) where TA acts as not only the building block but also the bioactive drug. We found that the TA gel can effectively regulate the microglial polarization towards anti-inflammatory phenotype when it was applied to microglia soon after oxygen-glucose deprivation (OGD), and anti-inflammatory microglia can then enhance the synaptic plasticity of neurons in vitro. Subsequently, the TA gel was implanted into the cavity of stroke mice induced by the photothrombotic (PT) method. The PT model can cause a cavity in the cortex, which reduces the difficulty of intracranial injection. The immunofluorescence (IF) stains suggest TA gel can also regulate the microglial polarization towards anti-inflammatory phenotype and further enhance neuroplasticity in vivo. The ethological tests including beam balance and rota-rod showed the significant recovery of motor function of stroke mice by TA gel. Additionally, the molecular biological analysis indicates TA gel plays an anti-inflammatory role and regulates the microglial polarization via the classical NF-κB signaling pathway. Consequently, TA gel shows promising potential in the treatment of subacute stroke to promote post-stroke rehabilitation due to its easy fabrication and significant therapeutic effect.

Materials

CMCS (MW 20 − 30 kDa, degree of carboxylation ∼80%, Beijing Solarbio Science & Technology Co., Ltd., CHN), TA (J&K Scientific Ltd., CHN), Cy5 (RuixiBio, CHN), phosphate buffered saline (PBS, Solarbio, CHN), normal saline (0.9%, China Resources Double-crane Pharmaceutical Co.,Ltd., CHN), paraformaldehyde (PFA, 4%, Solarbio, CHN), Sodium pentobarbital(Merck, Germany), rose bengal (Sigma-Aldrich, Germany).

Primary antibodies for IF staining:

rabbit anti-Iba-1 (ab178846, Abcam, UK), mouse anti-INOS (MA5-17139, Invitrogen), goat anti-CD206 (AF2535, R&D), mouse anti-PSD95 (ab2723, Abcam, UK), rabbit anti-VGlut1 (GTX133148, GeneTex, USA)

Primary antibodies for WB:

rabbit anti-CD206 (24595, CST, USA), rabbit anti-iNOS (GB113965, Solarbio, CHN), mouse anti-p65 (GB12142, Solarbio, CHN), rabbit anti-p-p65 (GB113882, Solarbio, CHN), rabbit anti-IĸBα (GB111509, Solarbio, CHN), rabbit anti-p-IĸBα (AF2002, AFFinity, USA), rabbit anti-Synaptophysin (GB11814, Solarbio, CHN), rabbit anti-PSD95 (GB11277, Solarbio, CHN), rabbit anti-CD16 (80006, CST, USA), rabbit anti-IL-1β (GB11113, Solarbio, CHN), rabbit anti-TGF-β (GB111876, Solarbio, CHN), rabbit anti-TLR4 (GB11519, Solarbio, CHN), rabbit anti-LaminB1 (GB111802, Solarbio, CHN), mouse anti-GAPDH (GB15002, Solarbio, CHN)

Preparation of the TA Gel

TA was dissolved in deionised water to obtain a TA solution at a concentration of 6 mg/mL. 50 mg CMCS was added into 500 uL TA solution to obtain a TA gel. The gel was centrifuged under 4000 rpm for 5 minutes to remove air bubbles.

In vitro Characterization of the TA Gel

Gelation

The gelation time of the TA gel was determined by inverted vial method. The TA solution was added to a small glass vial containing CMCS powder and mixed well to start the timer. 30 seconds later, the vial was inverted and the solution no longer flowed to prove that a gel had formed. The time taken for this process is the gelation time.

Morphological observation

The cross-sectional morphology of the TA gels was observed by scanning electron microscopy (SEM, Regulus S8230). The gel was freeze-dried, and then immersed in the liquid nitrogen and quickly cut for SEM observation. The samples were gold sprayed to improve the electrical conductivity of the samples prior to observation.

IR measurement

The as-prepared hydrogels were freeze-dried and characterized by FT-IR (Shimadzu IRTrace-100).

Degradation

The TA gel was freeze-dried and weighed and the mass was recorded as m₀. The TA gel was placed in a 10 mL centrifuge tube containing 8 mL of PBS solution. The centrifuge tubes were placed in a water bath shaker and gently shaken at 37°C. The sample was removed at the time interval required for the experiment, freeze-dried and the sample mass was weighed and recorded as m_x. The degradation rate (DR) of the TA gel was calculated by the following equation.

DR = (m₀ - m_x)/m₀✕100%

In vitro release of TA

The in vitro release of TA was measured by UV-Vis spectrophotometer (UV-1800). First, the absorbance of a series of TA solutions in a concentration gradient was measured to establish a standard curve (Figure S6). Then, 1 mL of TA gel was completely submerged in a 50 mL centrifuge tube containing 10 mL of PBS solution and gently shaken in a bath shaker at 37°C. 0.5 mL release solution was taken out at predetermined time pointed and supplemented with 0.5 mL fresh PBS solution. The solution was further measured by UV-Vis spectrophotometer (UV-1800) in order to get the in vitro release plot of TA.

In Vitro Cell Experiments

Biocompatibility of the TA gel

N2a cells were cultured with the medium extracted from TA gel in order to evaluate the biocompatibility of TA gel. 0.1 mL TA gel was prepared in the glass vial, and then 5 mL culture medium was added into the vial. The TA gel was extracted at 37°C for 24 h to obtain the TA gel extracted solution (TAG). N2a cells were cultured in 96-well plates (1 × 10⁴ cells/well) for 24 h. The medium was then replaced with different TAGs (0%, 50%, 100%) diluted by normal culture medium. Cells were cultured in a humidified environment at 37°C and 5% CO2 for 24h and 48h, respectively. After then, the cell viability was measured by live and dead cell assay according to the operation manual of the manufacture (CA1630, Solarbio, CHN) as well as CCK8 assay. The fluorescent images were acquired by fluorescence imaging microscopy (Olympus BX51, Japan).

The modulation of microglial phenotype by TA gel in vitro

BV2 Cell viability and cytotoxicity

BV2 (mouse microglial) was purchased from Wuhan Pronoxyl Life Sciences Co.. Cells were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (1 million U/L each) in a 37°C, 5% CO₂ incubator (SANYO (XD-101), Japan). Cells were cultured until they reached 70% or more confluence under the microscope, with the culture medium changed every two days and passaged every three days.

BV2 cells were seeded in 96-well plates (1×104 cells/well) and cultured with different TAGs (0%, 25%, 50%, 100%) for 24 h. After then, the cell viability was measured by CCK-8 assay and the cytotoxicity was measured by LDH assay.

Microglia polarization modulated by TA gel

BV2 cells were first cultured with a sugar-free medium and then placed in an OGD (glucose and oxygen deprivation) tank filled with gas (95% N2/5% CO₂) for 10 minutes. The tank with BV2 cells was closed and placed in the incubator for 3 hours without sugar and oxygen. Then, the OGD tank was removed and the sugar-free medium was changed with the sugar-containing medium. As soon as the replenishment of oxygen and glucose, the TAG (50%) was added. And BV2 cells were further cultured for 24h for examination.

The expressions of iNOS and CD206 in BV2 cells were firstly characterized by immunofluorescence staining (Olympus BX51, Japan). Furthermore, cells were collected and made into whole cell lysate. The expressions of CD16、CD206、IL-1β、TGF-β in BV2 cells were measured by western-blot (WB) method. Finally, the expressions of TLR4、phos-p65、p65、phos-IĸBα、IĸBα in BV2 cells were also measured by WB. Then, the cytoplasm and nucleus were separated and the expression of p65 in the cytoplasm and nucleus was measured by WB, respectively.

Synaptic plasticity modulated by BV2 cells in vitro

Four groups of BV2 cells including BV2, BV2 + 50%TAG, OGD BV2, OGD BV2 + 50%TAG, were cultured in the upper chamber of the transwell system for 12 h. The OGD operation lasted for 3 hours. After then, the culture mediums were removed and BV2 cells were washed by fresh medium thrice in order to completely remove the 50%TAG.

Two groups of N2a cells including N2a and OGD N2a were seeded on the lower plate of the transwell system and cultured in the mixing medium (BV2:N2a = 1:1). The OGD operation lasted for 3 hours. Four BV2 groups and two N2a groups were assembled in pairs to form eight complete transwell systems. The BV2 and N2a cells were co-cultured in the transwell systems for 24h to investigate the influences of BV2 cells on N2a cells. The viability was assessed by CCK-8 assay and the expression of synaptophysin and PSD-95 proteins was measured by WB.

In vivo Experiments

Animals

All in vivo studies were approved by the Animal Experiment Ethics Committee of Beijing Rehabilitation Hospital, Capital Medical University. All animal care, housing, surgical, and anesthetic procedures were performed in accordance with the Regulation for the Administration of Affairs Concerning Experimental Animals of China. Animals were housed five-per-cage at a standard temperature (22 ± 1℃) with ad libitum access to food and water. Male C57BL/6J mice (aged 12 weeks, 26.0 ± 1.5g,) were used in the experiments.

Photothrombotic Stroke Model

The mice were anaesthetized with isoflurane (4%, 3 min) and then the head was shaved and the scalp was sterilized. Then, they were fixed on a Mouse Stereotaxic and a midline incision was made along the rostrocaudal axis to separate the skull from the skin and expose the skull. The mice were injected with the rose red solution at a concentration of 10 mg/ml at a dose of 10 µl/g through the tail vein and circulated for 7 minutes. A laser with a wavelength of 561 nm and a power of 40 mW was used to irradiate the upper left side of the skull of mice for 15 minutes with a view to forming a thrombus. Mice's head skin was sutured and placed on a heating pad (37°C) for 5–10 min.

After the mice recovered from anesthesia, they were placed in a new cage and supplemented with food and water as needed. In this case, the sham-operated group was injected with the same dose of PBS solution through the tail vein and the rest of the steps were the same as above.

Injection of TA gel

At the 7th day after the stroke, all mice were divided into 5 groups based on ethological data. The five groups were: sham-operated (Sham), untreated (Stroke), TA gel (TAG), TA solution (TAS) and CMCS solution. Mice were anesthetized with isoflurane (4%, 3 min) and 3 µL of TA gel, TA solution and CMCS solution were injected into the cavity of the stroke model using a syringe, whereas the Sham and Stroke groups were left untreated.

In vivo imaging of TA gels

At the 7th day after stroke, 3 µL of Cy5-labelled TA gel was injected into the stroke cavity of the mice and photographed at the 7th, 9th, 14th, 17th and 21st day after stroke by a small animal fluorescence imaging system (IVIS® Lumina III, PerkinElmer, USA) to observe the degradation of the TA gel in the mice.

Ethology Assessment

Rotarod Test: Mice were trained on a Rotarod (Panlab Rotarod LE8505) at 10 rpm for 3 days prior to the former test. During the test, the speed of the Rotarod was accelerated uniformly from 4 rpm to 40 rpm over 5 minutes and the time when the mouse fell off the rotating axis was recorded.

Balance Beam Test: Motor coordination was quantified by counting the number of times that the right hind paw slid off the beam (approximately 80 cm long, 1.5 mm wide and 100 cm off the ground) during the experiment. Acquired training was performed for 3 days prior to the formal test.

The test was repeated three times for each mouse.

In vivo characterization of microglial polarization and neuroplasticity

On day 21 after stroke, 1% sodium pentobarbital was injected at a dose of 0.3mL to anaesthetize mice. As soon as exposing the heart, it was perfused with 0.9% saline from the left ventricle and then 4% PFA. The brain tissue was isolated and preserved in PFA (4%) for fixation. The microglia polarization in vivo was characterized by WB and IF staining, whereas the neuroplasticity in vivo was measured by WB, IF staining and Golgi staining (Servicebio, GP1152).

Brain tissue from the marginal zone of cortical brain infarction was collected and whole cell lysates were prepared. WB was performed to detect the expression of iNOS and CD206 to investigate the microglia polarization in vivo, synaptophysin and PSD-95 to explore synaptic plasticity in vivo, and phos-p65, p65, phos-IĸBα, IĸBα to demonstrate the role of NF-κB pathway in the microglia polarization.

3 mm thick slices of brain tissue were embedded in paraffin and 4 µm slices were cut out for subsequent experiments using a microtome (Leica RM2235, Germany). IF staining was performed by adding a primary antibody to the tissue sections and incubating overnight at 4°C. A secondary antibody working solution corresponding to the source of the primary antibody was then added and incubated for 30 min at 37°C, protected from light. nuclei were then stained with DAPI for 10 min (protected from light). Finally, fluorescence scanning of the slices was performed using the digital slice scanners (KF-PRO-020, KFBIO, Ningbo Jiangfeng Bioinformatics Co., Ltd.).

Statistics

For the immunofluorescence staining results, five fields of view (magnification 400) of the target area were selected for each image and the positive area was counted using Image J software. Statistical analysis was performed using GraphPad Prism software (version 5.01). Results were expressed as mean ± standard deviation (SD). Student’s T test was used to test the difference between two groups. One-way ANOVA (one-way analysis of variance) was used to test for multiple comparisons of differences between multiple groups. In all analyses, p < 0.05 was considered statistically significant, where *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 1 shows the reaction equation of the TA gel and the proposed mechanism of the TA gel that promotes poststroke rehabilitation. The hydrogel was formed by simply mixing CMCS and TA. Since TA usually acts as an H-donor and CMCS could be an H-receptor, the reaction can be carried out very fast under physiological pH 7.4 at body temperature without any external stimulus. TA is not only one necessary building block for hydrogel preparation, but also an effective drug to enhance neuroplasticity for poststroke rehabilitation. The TA gel can sustainably release TA to the peri-infarct area in vivo. It is proposed that TA as an anti-inflammatory drug can modulate the microglia polarization towards anti-inflammatory phenotype via classical NF-κB pathway. Subsequently, the anti-inflammatory microglia can play a vital role in enhancing neuroplasticity to promote functional recovery after stroke.

In vitro characterization of the TA gel was shown in Fig. 2. Figure 2a exhibited the successful formation of the TA hydrogel by the inverted vial method. Figure 2b showed the TA gel could be injected out from a syringe, indicating that it could be intracranially delivered into the brain by the syringe. The porous structure of the TA hydrogel was depicted by the SEM image in Fig. 2c. Figure 2d exhibited the FT-IR spectra of TA, CMCS and the TA gel. TA and CMCS showed strong, broad bands of − OH stretching vibration at 3480 and 3430 cm^− 1, respectively. In the spectrum of the TA gel, the − OH absorption peak shifted to 3410 cm^− 1, which demonstrated the presence of hydrogen bonds between them. The IR result indicated that TA gel is formed by the hydrogel bond interactions between TA and CMCS. The rheological properties of TA gel are shown in Fig. 2e. The low modulus ranging from 10³ to 10⁴ MPa makes the gel injectable to a certain extent ^[31]. The degradation and TA release behavior in vitro were shown in Fig. 2f and 2g. The weight loss plot of the TA gel during degradation exhibited the TA gel had gradually lost around 60% of weight for 16 days, whereas the in vitro release behavior showed TA can be sustainably released from the hydrogel for around two weeks. The biocompatibility and cytotoxicity of the TA gel is shown in Fig. 2h and Figure S1 by live/dead cell assay. The TA gel (TAG) was extracted by the culture medium, and the extracted culture medium was diluted by different volumes of culture medium and used to culture N2a cells. On the one hand, it shows the number of live cells in the different dilutions is similar to that cultured in the complete culture medium, indicating the satisfactory biocompatibility of the TA gel. On the other hand, both different dilutions and complete cell medium possessed very few dead cells, implying the low cytotoxicity of the TA gel. The biocompatibility of the TA gel is further characterized by the CCK assay in Fig. 2i, which is consistent with the results of the live/dead cell assay in Fig. 2h.

Figure 3a showed the schematics of BV2 cell culture. Figure 3b exhibited that cells treated with different concentrations of the extracted medium had a similar proliferation rate with cells treated with complete culture medium indicating good biocompatibility of the TA gel on BV2 cells. After OGD, the cell viability of BV2 cells in the control treated with a complete culture medium decreased sharply whereas the 50% TAG group significantly recover the viability of OGD BV2 cells. Figure 3c showed TAG groups also possessed similar low LDH release with the control implying the low cytotoxicity of the TA gel. Correspondingly, the LDH release in the control greatly increased after OGD, while the treatment of TAG significantly suppressed it. The IF images in Fig. 3d and Figure S2 depicted BV2 cells as the control expressed a high level of pro-inflammatory protein marker CD16 and a low level of anti-inflammatory protein marker CD206 after OGD. However, the TAG can significantly decrease the expression of CD16 and increase the expression of CD206 in OGD BV2 cells. The semi-quantitative results of IF images were shown in Fig. 3e and 3f, which clearly showed lower CD16 and higher CD 206 expression of the TAG group than the control after OGD with statistical differences. The WB strips and quantitative results were shown in Fig. 3g-3k, which showed similar results for IF stains. It also exhibited the TAG group possessed lower CD16 and higher CD 206 expression after OGD than the control with statistical differences. Thus, the above results clearly demonstrated our proposal that the TA gel can modulate BV2 cell polarization towards anti-inflammatory phenotype. Furthermore, as IL-1β and TGF-β are typical pro-inflammatory and anti-inflammatory factor secreted by BV2 cells, respectively, lower IL-1β expression and higher TGF-β expression in TAG group further implied the ability of TA gel to regulate anti-inflammatory polarization of the microglia. The N2A cells were co-cultured with BV2 cells in the transwell system as shown in Fig. 3l. The normal BV2 cells and OGD BV2 cells were firstly cultured with normal culture medium and TAG, respectively. Then, four groups including BV2, BV2 + TAG, OGD BV2, and OGD BV2 + TAG were co-cultured with normal N2A and OGD N2A, respectively. The WB strips and quantitative results were available in Fig. 3m-3o. It showed four groups cocultured with normal N2a cells didn’t show any statistical differences in the expression of synaptophysin and PSD95. However, significant differences were found for OGD N2A cells. It exhibited the lowest expression of two proteins when the OGD N2A was co-cultured with OGD BV2. This result indicated the stroke significantly leads to the apoptosis of neurons in vitro as synaptophysin and PSD95 are two major proteins involving synaptic plasticity. When the OGD N2A cells were co-cultured with OGD BV2 + TGA, the expression of synaptophysin and PSD95 significantly increased compared with those with OGD BV2. Additionally, BV2 + TAG can also make OGD N2A cells express more synaptophysin and PSD95 than BV2. These results implied that TA gel can exert a beneficial influence on enhancing neuroplasticity in vitro.

Figure 4a depicted the protocol of the in vivo test in this study. The TA gel is implanted into the infarct cavity of the stroke mouse at the 7th day after the stroke induced by the PT model. The ethological tests are carried on at the 6th, 8th, 11th, 14th, and 21st day in order to observe the recovery of motor function of the stroke mouse by TA gel. The mouse was sacrificed at the 21st day for WB and IF measurement. Figure 4b showed the in vivo images of the implanted gel. As shown in Figure S3, the fluorescent molecule, Cy5 was covalently bonded to the TA gel thus it could be visually detected by a small animal fluorescence imaging system. The immunofluorescence spectra and the SEM image of the fluorescence TA gel were shown in Figure S4 and Figure S5, respectively. As soon as the fluorescence TA gel was implanted in the cavity, a strong fluorescence signal could be observed indicating the successful intracranial delivery of the gel. The intensity of the signal gradually weakened with the implantation time, implying the gradual degradation of the gel in vivo. The signal was still present at the 18th day whereas it could hardly be detected at the 21st day. This implied the TA gel can stay at least 12 days in vivo to sustainably release tannic acid to enhance the neuroplasticity. Figure 4c-4e tentatively investigated the ability of TA gel to regulate the polarization of microglia in vivo. The sham group has a relative low level of both iNOS and CD206 expression as the microglia don’t polarize in the healthy mouse, whereas stroke mice exhibit the highest iNOS and the lowest CD206 expression indicating the polarization of the microglia towards pro-inflammatory phenotype. Nevertheless, both TA solution (TAS) and TA gel (TAG) groups possess lower iNOS and higher CD206 expression than the stroke group with statistical differences, implying the tannic acid has the ability to regulate the polarization of the microglia towards anti-inflammatory phenotype. Between the two TA groups, TAG has even better performance to regulate the polarization of the microglia than TAS, hinting that the TA gel has the promising potential to enhance neuroplasticity and recover the motor function of stroke mice. However, the CMCS solution group has the similar iNOS and CD206 expression with the stroke group and didn’t show any ability to modulate microglia to express anti-inflammatory phenotype. Figure 4f and 4g exhibited results of the balance beam and rotarod measurement in order to evaluate the recovery of motor function of stroke mice by TA gel. In the beam balance test, stroke sharply increases the foot falling number of mice, while the treatment of TA gel can gradually decrease the falling number with the treatment time. Although it has a similar baseline with other groups including the stroke, the TAS and the CMCS groups before receiving gel administration at the 6th day, the TAG group begins to show statistical differences with other groups since11st day and approaches that of the sham group at the 21st day. The TAS group also has certain positive influences on decreasing the falling number, whereas the CMCS group doesn’t show any positive influences. In the rota-rod test, the opposite tendency was observed. The stroke greatly decreases the latency to fall of mice, while the treatment of TA gel can gradually increase it with the treatment time. The TAG group also begins to show statistical differences with other groups since the 11th day. At the 21st day, the latency to fall of the TAG has closely approached that of the sham group, and no statistical difference is found between them. The TAS group also has positive influences to some extent, whereas the CMCS group doesn’t show any positive influences. Consequently, these ethological results indicated the satisfactory recovery of motor function of stroke mice by TA gel.

Figure 5 showed the images of IBA-1/iNOS and IBA-1/CD206 staining of the peri-infarct area in stroke mice. IBA-1 is the specific marker of the activated microglia, thus the colocalization of IBA-1⁺iNOS⁺ represents the pro-inflammatory microglia whereas that of IBA-1⁺CD206⁺ corresponds to the anti-inflammatory microglia. It can be seen in Fig. 5a that the IBA-1 expression is very weak in the sham group as most of the microglia remains at rest in the healthy mouse. It becomes much stronger after stroke indicating the activation of microglia in the peri-infarct area. It seems the TAG group expresses a slightly lower IBA-1 expression than the TAS and the control, showing that the TAG group can suppress the activation of the microglia to some extent. Furthermore, the semi-quantitative results of the colocalization of IBA-1⁺iNOS⁺and IBA-1⁺CD206⁺ were depicted in Fig. 5c and 5d. It exhibited the stroke group expresses the highest colocalization of IBA-1⁺iNOS⁺ and the lowest colocalization of IBA-1⁺CD206⁺, implying that the microglia in this group mainly express the pro-inflammatory phenotype. On the contrary, the TAG group possesses the lowest colocalization of IBA-1⁺iNOS⁺and the highest colocalization of IBA-1⁺CD206⁺, indicating that the microglia in the peri-infarct area have preferably polarized towards anti-inflammatory phenotype in this group. The colocalization of both IBA-1⁺iNOS⁺ and IBA-1⁺CD206⁺ of the TAS group is in the middle, showing TAS has a little effect on modulating microglia polarization.

Figure 6a-6d evaluated the synaptic plasticity of stroke mice by the double staining of PSD95 and Vglut1. Since PSD95 and Vglut1 are typical postsynaptic and presynaptic markers, respectively, the colocalization of PSD95⁺Vglut1⁺ can indicate the synaptic connection. Spontaneously, better synaptic connection means enhanced synaptic plasticity which may further lead to better functional recovery. A similar tendency was found for PSD95 and Vglut1. The sham group has the highest PSD95 and Vglut1 expression indicating the perfect synaptic networks in the healthy mouse. The TAG group possesses the second place implying the enhancement of the synaptic plasticity by the TA gel after stroke. The TAS is in the third, showing that the TA solution has a little positive influence on enhancing the synaptic plasticity. Not surprisingly, the stroke group showed the lowest PSD95 and Vglut1 expression due to the neuron apoptosis caused by the ischemic stroke. Furthermore, the colocalization of PSD95⁺Vglut1⁺ showed the same trend with PSD95 and Vglut1 expressions that the sham group has the best synaptic connection and the TAG is the next. The TAS and stroke possess the third and last place. Figure 6e-6g showed the WB results of synaptophysin and PSD-95 expressions in the peri-infarct zone. Synaptophysin and PSD-95 are two representative proteins related to synaptic plasticity. It showed the sham group has the highest expression for these two proteins and the TAG is the second. The TAS and stroke possess the last two places in turn. Hence, the colocalization of PSD95⁺Vglut1⁺ as well as the WB results of synaptophysin and PSD-95 expressions demonstrated the TA gel treatment can effectively enhance synaptic plasticity in vivo.

After the ischemic stroke, the cortex adjacent to the infarct cavity undergoes morphological and functional remodeling including dendritic spine density and dendritic branching, which is important to form new neural connections. To investigate the effects of the TA gel treatment on dendritic spine structure after ischemic stroke, we performed Golgi silver impregnation to detect neuronal dendritic spines in the cortical peri-infarct area in sham, stroke, and TAG-treated mice in Fig. 7. We found that PT stroke induced a significant reduction in the number of dendritic spines and branch numbers in Fig. 7b and 7c. Interestingly, the TA gel treatment remarkably increased the dendritic plasticity compared with the stroke group, indicating TA gel treatment can effectively enhance dendritic plasticity in vivo.

TLR4/NF-κB signaling plays an essential regulatory role in stroke-induced pro-inflammatory microglial polarization^{[15, 32, 33]}, we tested whether TA gel regulated the anti-inflammatory microglial polarization by inhibiting the activation of TLR4/NF-κB signaling pathway in PT stroke model in vitro and in vivo studies. The protein expression of TLR4 (Fig. 8a and 8b), phosphorylated IKBα (p-IKBα) (Fig. 8a and 8e), phosphorylated p65 (p-p65) (Fig. 8a and c) were markedly increased in the OGD stimulated group compared to the control group. However, these protein expressions were significantly decreased in OGD-challenged BV2 cells after being treated with TA gels. Furthermore, TA gel inhibited OGD-induced increase in the ratio of p-IKBα/IKBα and p-p65/p65. No significant changes in the protein expressions of p65 and IKBα were observed in these groups.

To investigate the nuclear translocation of NF-κB p65 in BV2 microglia cells after OGD stimulation, we measured the protein levels of p65 in the nucleus and cytoplasm of OGD BV2 cells, respectively (Fig. 8i-8k). TA gel treatment dramatically inhibited OGD-induced elevation of p65 in nucleus, but enhanced the p65 expression in the cytoplasm. These data reveal that TA gel abolished OGD-induced nucleus translocation of p65 from the cytoplasm to the nucleus. Overall, the in vitro results indicate that the inhibitory effect of TA gel on pro-inflammatory microglial polarization is associated with suppressing the activation of the TLR4/NF-κB signaling pathway after stroke.

This hypothesis was further supported by in vivo studies examining the expressions of NF-κB related proteins in PT mice brain. Consistent with in vitro results, TA gel treatment dramatically inhibited the phosphorylation of p65 and IKBα, and reduced the ratio of p-p65/p65 and p-IKBα/IKBα in the peri-infarct cortex (Fig. 8l-8r) at 21 days after PT stroke. These findings support that TA gel dramatically inhibited NF-κB activation in BV2 microglia after PT stroke.

Historically, tannin-containing plants have been used by both Western and Eastern cultures as traditional medicine for various disorders, in particular as an antiphlogistic for inflammatory conditions.^{[34, 35]} Recently, it has been reported that TA can significantly reduce behavioral impairment, oxidative damage, and inflammatory responses caused by traumatic brain injury.^[36] Ashabi, et, al. also found long-term treatment with TA improves hypoperfusion-induced motor deficits and memory dysfunction in a rat model of unilateral common carotid artery occlusion (UCCAO).^[37] Spontaneously, we proposed in Fig. 1 that TA is able to regulate the polarization of microglia by exerting its anti-inflammatory properties.

As mentioned above, an injectable TA gel is the best carrier for TA administration as it can sustainably release TA in the infarct cavity to regulate microglia polarization on site. The successful formation of the hydrogel is demonstrated by the inverted vital result in Fig. 2a. According to our previous work^[38], the rheological property of the TA gel can be modulated by the feed ratio of TA and CMCS. Thus, we adopted a suitable feed ratio in this study to assure the injectability of the TA gel according to our experiences. Figure 2b demonstrated the TA gel can be smoothly injected from a syringe, so that it can meet the requirement of intracranial delivery in this study. The degradation plot in Fig. 2e showed the TA gel lost 60% of weight within 16 days. According to the animal test protocol in Fig. 4a, the TA gel injection is arranged at the 7th day after PT-induced stroke and the sacrifice is at the 21st day, thus the TA gel is expected to be able to stay in vivo for around two weeks. The in vitro degradation behavior in Fig. 2e indicated the TA gel can meet this requirement. As discussed above, TA was spontaneously released with the degradation of the TA gel. The in vitro release behavior was shown in Fig. 2f. Similarly, Fig. 2f also showed the TA gel can assure a sustainable release of TA within around two weeks to meet the requirement of the animal test protocol.

Figure 2g and 2h demonstrated that the hydrogel shows high biocompatibility and low cytotoxicity cultured with N2A cells. Furthermore, Fig. 3a also shows it also has high biocompatibility and low cytotoxicity cultured with BV2 cells by CCK8 and LDH assays. Since neurons and microglia are two major cells in the brain, these results indicate the TA gel could be used in the brain without any risks. Many literatures reported the anti-inflammatory ability of TA. Parvez, et.al intraperitoneally injected TA solution into a TBI mouse and found TA can effectively downgrade the expression of IL-β (Interleukin-1 beta) and TNF-ɑ.^[36] As IL-β is a typical pro-inflammatory cytokine secreted by microglia, the results in the literature imply TA is able to stimulate microglia to decrease the secretion of pro-inflammatory cytokines and polarize towards anti-inflammatory phenotype. Furthermore, Ashabi reported oral administration of TA solution can also downgrade the expression of NF-κB and TNF-ɑ in UCCAO rats.^[37] Since the NF-κB pathway is a classic inflammatory-related pathway and the suppression and inhabitation of the NF-κB pathway can significantly suppress the local inflammatory response. The IF stains and WB results in Fig. 3d-3k clearly showed the TAG can significantly decrease the CD16 expression and increase the CD206 expression in the OGD BV2 cells in vitro, exhibiting the potential to alter the activated microglial phenotype in the stroke mouse. Furthermore, the WB results in Fig. 3h, 3i and 3k showed the OGD microglia treated with TAG secrets less pro-inflammatory IL-β and more anti-inflammatory TGF-β than that without TAG. The phenotype of the microglia plays a vital role in neuroplasticity. The pro-inflammatory microglia produce destructive cytokines, exaggerating brain damage. In contrast, anti-inflammatory microglial release trophic factors, promoting tissue repair.^[39] Fig. 3l-3p clearly showed the viability as well as the expression of synaptic plasticity-related proteins such as synaptophysin and PSD95 of OGD N2A cells were significantly upgraded by anti-inflammatory microglia that are regulated by TAG. Thus, it demonstrated TA gel is able to enhance neuroplasticity in vitro by regulating microglia to polarize towards an anti-inflammatory phenotype.

Figure 4b shows the in vivo images of TA gel with different implantation time points. Fluorescence imaging of the mouse brain implied the presence of TA gel in the mouse brain. The presence of the hydrogel in vivo during the treatment course is the prerequisite for maintaining the local concentration of TA. As the fluorescence intensity decreased with the implantation time, the implanted TA gel gradually degraded in vivo. As TA acts as both a building block and a bioactive drug, the degradation of TA gel would release TA simultaneously. The gradual decrease of fluorescence signal also indicated the sustainable release of TA from the TA gel, which is expected to be able to regulate microglial polarization in vivo. Five groups including sham, stroke, CMCS, TAS and TAG were evaluated by WB measurement to investigate their influences on the in vivo microglia polarization in Fig. 4c and 4d. It showed the CMCS has the same performance as the stroke group and doesn’t show any ability in regulating microglia polarization. Both TAG and TAS exhibited the ability to modulate microglia to express anti-inflammatory phenotype after stroke, indicating TA is the active ingredient in the TA gel to regulate microglia polarization. Furthermore, it also clearly showed TA gel has a better ability to regulate microglia polarization than TA solution. In our previous work,^[40] we prepared a multifunctional hydrogel loaded with BDNF (brain-derived neurotrophic factor) and VEGF (vascular endothelial growth factor) for post-stroke rehabilitation. It was also found that the multifunctional hydrogel always had better performances than the solution of BDNF and VEGF in regulating microglia polarization as well as enhancing angiogenesis and neuroplasticity because the sustainable release behavior of the multifunctional hydrogel can maintain a high growth factor concentration in the peri-infarct area. Similarly, TA gel also has a much better ability to regulate microglia polarization than TA solution due to this reason in this study. The ethological measurement in Fig. 4e and 4f showed the same tendency as the WB results in Fig. 4c and 4d. As the microglial polarization towards anti-inflammatory phenotype can enhance neuroplasticity, the consistent results between ethology and microglia polarization are quite reasonable. Moreover, both the balance beam and rotarod showed that the TA gel exhibited better ethological results than the TA solution with a statistical difference since the 11th day, indicating better functional recovery of TA gel-treated stroke mice. Certainly, better functional recovery also results from the sustainable release of TA in the peri-infarct area. Additionally, since both ethology and WB measurements showed CMCS has no influence on microglia polarization and subsequent functional recovery, the CMCS solution group wasn’t investigated anymore in the following in vivo measurements.

The microglial polarization was further investigated by IF stains in Fig. 5. As resident immune cells in the central nervous system, microglia’s primary function is to maintain normal immune homeostasis. According to the pathology of the stroke, they are activated soon after stroke and dynamically polarize from a beneficial anti-inflammatory phenotype toward a neurotoxic pro-inflammatory phenotype accompanied by morphological and functional changes. At the subacute and chronic stages, microglia mainly possess the pro-inflammatory phenotype to release pro-inflammatory cytokines, which exert a detrimental influence on neuroplasticity, such as synaptic remodeling, axonal sprouting, and dendritic spine regeneration. Therefore, strategies aimed at driving anti-inflammatory microglial polarization have emerged as a potential rehabilitative approach to restore neurofunction after stroke. The activation of microglia was clearly demonstrated in Fig. 5 as the stroke, the TAS and the TAG groups all possessed higher IBA-1 expression than the sham group. However, the TAG exhibited lower IBA-1 expression than the stroke and the TA solution groups, indicating TA gel treatment can suppress microglial activation after stroke. Furthermore, it can be found that the TAG showed the lowest percentage of iNOS⁺IBA-1⁺ among total IBA-1⁺microglia and the highest percentage of CD206⁺IBA-1⁺microglia among total IBA-1⁺ among all the groups, indicating that there were the fewest pro-inflammatory microglia and the most anti-inflammatory microglia in the peri-infract zone of mice treated with TA gel. As mentioned above, these anti-inflammatory microglia can greatly enhance neuroplasticity to promote poststroke rehabilitation. Furthermore, the TAG was shown to have lower iNOS/IBA-1 and higher CD206/IBA-1 colocalization than the TAS group, which again demonstrated the advantage of TA’s sustainable release behavior in TA gel. In addition, microglia were proved to be involved in synaptic plasticity through releasing pro-/anti-inflammatory factors or synaptic pruning after stroke. Anti-inflammatory microglia are known to secret neuroprotective factors including BDNF. Thus, shifting pro-inflammatory microglia to an anti-inflammatory phenotype can enhance synaptic plasticity and promote functional outcomes following stroke. Therefore, it is believed that the TA gel can indirectly enhance synaptic plasticity by modulating microglial polarization towards anti-inflammatory phenotype.

Synaptic plasticity plays a vital role in poststroke rehabilitation, as it is directly related to the function recovery after stroke.^[41] PSD-95 and Vglut1 double staining was used to investigate synaptic plasticity in the peri-infarct area in this study. PSD-95 is the most important and abundant scaffold protein in the postsynaptic membrane, ^{[27, 42]} whereas Vglut1 is commonly used as a presynaptic marker. ^{[43, 44]} Thus, the connection of these two proteins represents the enhancement of synaptic plasticity. ^[45–47] As shown in Fig. 6a-6d, the amounts of both PSD-95 and Vglut-1 sharply decreased in stroke mice, which indicated the loss and apoptosis of the cranial nerve due to ischemic onset. Previous studies have demonstrated that the anti-inflammatory microglia can improve synaptic plasticity and motor function in a stroke model. Here, the positive puncta of these two proteins in the peri-infract area gradually recovered in both TAG and TAS groups. As discussed in above, TA treatment can effectively regulate microglial to express anti-inflammatory phenotype, this result supported our previous conclusion that the anti-inflammatory microglia can improve synaptic plasticity. Furthermore, mice treated with the TAG exhibited more positive particles of the two proteins than that treated with TAS. Thus, the TA gel possessed a better ability to enhance poststroke synaptic plasticity. Moreover, as the colocalization of PSD-95 and Vglut1 reflects the synaptic connection, it can better characterize synaptic plasticity in the peri-infarct area. It was found in Fig. 6 that the colocalization of PSD-95 and Vglut1 was markedly decreased after the stroke and recovered to some extent after treatment with both TAG and TAS. The TA gel treatment still induced more synaptic connections than the TA solution. It was reported in many literatures that the synaptic connection is directly related to motor functions. We also observed in our previous work that the increasing of the synaptic connections can promote functional recovery of stroke mice. Thus, the best synaptic connection of the TAG in Fig. 6 supported the ethological results in Fig. 4 that TAG has the best functional recovery after stroke. Furthermore, it also showed that sustainable release of TA in vivo by the TAG can achieve better synaptic connection than one-time intracranial administration by the TAS group. The WB results in Fig. 6e-6g further demonstrated that the TAG has the best ability to enhance synaptic plasticity since it possessed the highest synaptophysin and PSD95 expression in vivo. Hence, based on the above results of PSD-95 and Vglut1 co-staining as well as the WB results of synaptophysin and PSD95 expressions, it can be concluded that the TA gel can promote synaptic plasticity by sustainably releasing TA to regulate microglia polarization toward anti-inflammatory phenotype in vivo.

Dendritic spine densities, an independent line of evidence for poststroke recovery, are one of the neural structural and functional plasticity recognized as the cornerstone of functional restoration after stroke.^[48] Dendritic plasticity plays a crucial role in neural network rebuilding in the peri-infarct area. The increase in dendritic spine densities occurs in the surviving neurons next to the infarct cavity and contributes to rebuilding neural networks taking over the function of the lost neurons.^[49] Here, we detected significant changes in dendritic spine densities in the peri-infarct area in the cortex based on Golgi staining in Fig. 8. After PT stroke, dendritic spine density in the peri-infarct region is greatly reduced, whereas the TA gel treatment remarkably reversed the stroke-induced decrease in dendritic spine density. Thus, TA gel-afforded functional restoration might be partially attributed to dendritic spine rebuilding. In line with our results, several studies have demonstrated that poststroke functional outcomes were associated with the increases in dendritic spine density.^{[50, 51]} Consequently, it can be concluded that TA gel treatment after stroke can significantly enhance neuroplasticity in the peri-infarct zone as the PSD-95 and Vglut1 co-staining as well as the WB results of synaptophysin and PSD95 proved the enhancement of synaptic plasticity and the Golgi staining demonstrated the improvement of dendritic plasticity.

Several transcription factors such as STAT1,^[15] NF-κB p65,^[14] IRF5,^[52] were demonstrated to be involved in pro-inflammatory microglial polarization after stroke. The therapeutic approaches inhibiting the activation of these transcription factors to suppress pro-inflammatory microglial polarization might provide a novel restorative strategy for stroke. Zhao et al. designed a nanoparticle (NP) in which Ferulic acid diacid with an adipic acid linker (FAA) and tannic acid (TA) were used as shell and core molecules, respectively.^[53] They found that NP treatment significantly inhibited NF-κB activation, and reduced Iba-1 expression in α-synuclein-stimulated microglia. Furthermore, the anti-inflammatory effects of TA were associated with the suppression of NF-κB pathway activation in LPS-induced BV2 microglial cells.^[54] In this study, we have indicated that TA gel shifted microglial polarization towards an anti-inflammatory phenotype following PT stroke. The mechanisms underlying TA gel-mediated microglial polarization after stroke remain to be elucidated. Our western blot data showed that TA gel decreased the protein expression of TLR4 and NF-κB related proteins including p-IKBα and p-p65 in OGD-induced microglia cells. Also, TA gel administration reduced the translation of NF-κB p65 from cytoplasm to nucleus; thereby alleviating pro-inflammatory microglial polarization. Our findings demonstrated that TA gel switched pro-inflammatory microglia phenotype to an anti-inflammatory state at least partially through blocking NF-κB activation.

TA can act as both a building block to form an injectable TA gel with CMCS and a bioactive drug to promote post-stroke rehabilitation in this study. It showed the TA gel can regulate OGD BV2 cells to polarize towards anti-inflammatory phenotype in vitro. Then, the expression of synaptophysin and PSD95 in the OGD N2a cells was effectively recovered by TA gel-treated OGD BV2 cells, implying the ability of the TA gel to enhance in vitro neuroplasticity by modulating microglia polarization. After being injected into the stroke cavity of mice, the ability of the TA gel to modulate microglia polarization and subsequently enhance neuroplasticity was further proved by IF stains, WB as well as Golgi staining. The recovery of the motor function of stroke mice was illustrated by the ethological measurements including rota-rod and balance beam. Additionally, it was proven both in vitro and in vivo that the TA gel modulates microglia to polarize towards anti-inflammatory phenotype via NF-κB pathway. Consequently, it shows the injectable TA gel can promote post-stroke rehabilitation of stroke mice through modulating microglial polarization to enhance neuroplasticity.

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Competing interests

The authors declare no competing financial interest.

Funding

This work is financially supported by the National Natural Science Foundation of China (82271325, 82272616, 82102655), Beijing Municipal Natural Science Foundation (7222102), and the R&D Program of Beijing Municipal Education Commission (KM202210025001).

Authors’ contributions

These authors contributed equally: Z. Liu, S. Zhang, and Y. Ran

Conceptualization: Z. Liu, H. Geng, and S. Zhang; Methodology: Z. Liu and F. Gao; Investigation: S. Zhang and Y. Ran; Analysis, visualization, and validation: S. Zhang and Y. Ren; Resources: F. Gao and G. Tian; Writing – original draft: Z. Liu and S. Zhang; Writing – review & editing: L. Ye and H. Geng; Supervision and funding acquisition: L. Ye, W. Su, J. Xi, and Z. Feng. The authors read and approved the final manuscript.

Acknowledgments

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Tannic acid as building block constructing injectable hydrogel and regulating microglial phenotype to enhance neuroplasticity for post-stroke rehabilitation

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Abstract

Background

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Results

Conclusion

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Introduction

Materials and Methods

Materials

Preparation of the TA Gel

In vitro Characterization of the TA Gel

Gelation

Morphological observation

IR measurement

Degradation

In vitro release of TA

In Vitro Cell Experiments

Biocompatibility of the TA gel

The modulation of microglial phenotype by TA gel in vitro

BV2 Cell viability and cytotoxicity

Microglia polarization modulated by TA gel

Synaptic plasticity modulated by BV2 cells in vitro

In vivo Experiments

Animals

Photothrombotic Stroke Model

Injection of TA gel

In vivo imaging of TA gels

Ethology Assessment

In vivo characterization of microglial polarization and neuroplasticity

Statistics

Results

Discussion

Conclusion

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