Nanoarchitectonics of tannic acid based injectable hydrogel regulate the microglial phenotype to enhance neuroplasticity for poststroke rehabilitation.

BACKGROUND: Stroke is the second leading cause of mortality and disability worldwide. Poststroke rehabilitation is still unsatisfactory in clinics, which brings great pain and economic burdens to stroke patients. In this study, an injectable hydrogel in which tannic acid (TA) acts as not only a building block but also a therapeutic drug, was developed for poststroke 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 properties, and TA release behavior of the hydrogel were characterized. The abilities of the TA gel to modulate microglial (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 stroke mice to evaluate motor function recovery, microglial polarization, and neuroplasticity in vivo. The molecular pathway through which TA modulates microglial polarization was also explored both in vitro and in vivo. RESULTS: The TA gel exhibited sustainable release behavior of TA. The TA gel can suppress the expression of CD16 and IL-1ß, and upregulate the expression of CD206 and TGF-ß in oxygen and glucose-deprived (OGD) BV2 cells, indicating the regulation of OGD BV2 cells to an anti-inflammatory phenotype in vitro. This finding further shows that the decrease in synaptophysin and PSD95 in OGD N2a cells is effectively recovered by anti-inflammatory BV2 cells. Furthermore, the TA gel decreased CD16/iNOS expression and increased CD206 expression in the peri-infarct area of stroke mice, implying anti-inflammatory polarization of microglia in vivo. The colocalization of PSD95 and Vglut1 stains, as well as Golgi staining, 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 that the TA gel regulated microglial 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 poststroke rehabilitation in the clinic.

Design and Fabrication of Polymeric Hydrogel Carrier for Nerve Repair.

Nerve regeneration and repair still remain a huge challenge for both central nervous and peripheral nervous system. Although some therapeutic substances, including neuroprotective agents, clinical drugs and stem cells, as well as various growth factors, are found to be effective to promote nerve repair, a carrier system that possesses a sustainable release behavior, in order to ensure high on-site concentration during the whole repair and regeneration process, and high bioavailability is still highly desirable. Hydrogel, as an ideal delivery system, has an excellent loading capacity and sustainable release behavior, as well as tunable physical and chemical properties to adapt to various biomedical scenarios; thus, it is thought to be a suitable carrier system for nerve repair. This paper reviews the structure and classification of hydrogels and summarizes the fabrication and processing methods that can prepare a suitable hydrogel carrier with specific physical and chemical properties. Furthermore, the modulation of the physical and chemical properties of hydrogels is also discussed in detail in order to obtain a better therapeutic effect to promote nerve repair. Finally, the future perspectives of hydrogel microsphere carriers for stroke rehabilitation are highlighted.

Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF-κB Suppression and NLRP3 Inflammasome Inhibition.

NLRP3 inflammasome-mediated pyroptosis is a proinflammatory programmed cell death pathway, which plays a vital role in functional outcomes after stroke. We previously described the beneficial effects of curcumin against stroke-induced neuronal damage through modulating microglial polarization. However, the impact of curcumin on microglial pyroptosis remains unknown. Here, stroke was modeled in mice by middle cerebral artery occlusion (MCAO) for 60 minutes and treated with curcumin (150 mg/kg) intraperitoneally immediately after reperfusion, followed by daily administrations for 7 days. Curcumin ameliorated white matter (WM) lesions and brain tissue loss 21 days poststroke and improved sensorimotor function 3, 10, and 21 days after stroke. Furthermore, curcumin significantly reduced the number of gasdermin D+ (GSDMD+) Iba1+ and caspase-1+Iba1+ microglia/macrophage 21 days after stroke. In vitro, lipopolysaccharide (LPS) with ATP treatment was used to induce pyroptosis in primary microglia. Western blot revealed a decrease in pyroptosis-related proteins, e.g., GSDMD-N, cleaved caspase-1, NLRP3, IL-1ß, and IL-18, following in vitro or in vivo curcumin treatment. Mechanistically, both in vivo and in vitro studies confirmed that curcumin inhibited the activation of the NF-κB pathway. NLRP3 knocked down by siRNA transfection markedly increased the inhibitory effects of curcumin on microglial pyroptosis and proinflammatory responses, both in vitro and in vivo. Furthermore, stereotaxic microinjection of AAV-based NLRP3 shRNA significantly improved sensorimotor function and reduced WM lesion following curcumin treatment in MCAO mice. Our study suggested that curcumin reduced stroke-induced WM damage, improved functional outcomes, and attenuated microglial pyroptosis, at least partially, through suppression of the NF-κB/NLRP3 signaling pathway, further supporting curcumin as a potential therapeutic drug for stroke.

Baicalein ameliorates ischemic brain damage through suppressing proinflammatory microglia polarization via inhibiting the TLR4/NF-κB and STAT1 pathway.

Microglial polarization mediated neuroinflammation plays an important role in the pathological process of stroke. The aim of this study is to determine whether baicalein indirectly ameliorates neuronal injury through modulating microglial polarization after stroke and if so, then by what mechanism. The effects of baicalein on microglial polarization were revealed through the middle cerebral artery occlusion mouse model (MCAO, n = 6), the lipopolysaccharide (LPS) + interferon-γ (IFN-γ) and oxygen-glucose deprivation (OGD) induced neuroinflammatory microglia model (BV2, n = 3), respectively. Mice were treated with baicalein (100 mg/kg, i.g.) after reperfusion, and followed by daily administrations for 3 days. Results showed that the infarct volumes at 3 d in vehicle and baicalein-treated MCAO mice were 91.18 ± 4.02% and 55.36 ± 4.10%. Baicalein improved sensorimotor functions (p < 0.01) after MCAO. Real-time PCR revealed that baicalein decreased proinflammatory markers expression (p < 0.05), while elevated the anti-inflammatory markers (p < 0.05) in vivo and in vitro. Both western blot and immunofluorescent staining further confirmed that baicalein reduced proinflammatory marker CD16 levels (p < 0.01) and enhanced anti-inflammatory marker CD206 or Arg-1 levels (p < 0.05). Notably, baicalein suppressed the release of proinflammatory cytokines (p < 0.05) and nitric oxide (NO, p < 0.001). Mechanistically, baicalein prevented increases in TLR4 protein levels (p < 0.001), the phosphorylation of IKBα and p65 (p < 0.01), and the nuclear translocation of NF-κB p65 (p < 0.05). The NF-κB inhibitor, BAY 11-7085, enhanced the inhibitory effect of baicalein on the proinflammatory microglial polarization. Baicalein also inhibited the phosphorylation of signal transducer and activator of transcription 1 (STAT1, p < 0.001). A microglia-neuron co-culture system revealed that baicalein driven neuroprotection against OGD induced neuronal damage through modulating microglial polarization (p < 0.05). Baicalein indirectly ameliorates neuronal injury after stroke by polarizing microglia toward the anti-inflammatory phenotype via inhibition of the TLR4/NF-κB pathway and down-regulation of phosphorylated STAT1, suggesting that baicalein might serve a potential therapy for stroke.

Melatonin Protects Against Ischemic Brain Injury by Modulating PI3K/AKT Signaling Pathway via Suppression of PTEN Activity.

Stroke is one of the leading causes of death and disability worldwide with limited therapeutic options. Melatonin can attenuate ischemic brain damage with improved functional outcomes. However, the cellular mechanisms of melatonin-driven neuroprotection against post-stroke neuronal death remain unknown. Here, distal middle cerebral artery occlusion (dMCAO) was performed in C57BL/6j mice to develop an ischemic stroke in vivo model. Melatonin was injected intraperitoneally immediately after ischemia, and 24 and 48 hours later. Melatonin treatment, with 5 to 20 mg/kg, elicited a dose-dependent decrease in infarct volume and concomitant increase in sensorimotor function. At the molecular level, phosphorylation of PTEN and Akt were increased, whereas PTEN activity was decreased in melatonin treated animals 72 hours after dMCAO. At the cellular level, oxygenglucose deprivation (OGD) challenge of neuronal cell line Neuro-2a (N2a) and primary neurons supported melatonin's direct protection against neuronal cell death. Melatonin treatment reduced LDH release and neuronal apoptosis at various time points, markedly increased Akt phosphorylation in neuronal membrane, but significantly suppressed it in the cytoplasm of post-OGD neurons. Mechanistically, melatonin-induced Akt phosphorylation and neuronal survival was blocked by Wortmannin, a potent PIP3 inhibitor, exposing increased PI3K/Akt activation as a central player in melatonin-driven neuroprotection. Finally, PTEN knock-down through siRNA significantly inhibited PI3K/Akt activation and cell survival following melatonin treatment, suggesting that melatonin protection against ischemic brain damage, is at least partially, dependent on modulation of the PTEN/PI3K/Akt signaling axis.

Melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti-inflammatory phenotype through STAT3 pathway.

AIMS: Microglia and infiltrated macrophages play important roles in inflammatory processes after ischemic stroke. Modulating microglia/macrophage polarization from pro-inflammatory phenotype to anti-inflammatory state has been suggested as a potential therapeutic approach in the treatment of ischemic stroke. Melatonin has been shown to be neuroprotective in experimental stroke models. However, the effect of melatonin on microglia polarization after stroke and underlying mechanisms remain unknown. METHODS: In vivo, cerebral ischemia was induced by distal middle cerebral artery occlusion (dMCAO) in C57BL/6J mice. Melatonin was injected intraperitoneally (20 mg/kg) at 0 and 24 hours after ischemia. In vitro, the microglial cell line BV2 was stimulated to the pro-inflammatory state with conditioned media (CM) collected from oxygen-glucose deprivation (OGD) challenged neuronal cell line Neuro-2a (N2a). Real-time PCR was utilized to detect the mRNA expression of microglia phenotype markers. Activation of signal transducer and activator of transcription 3 (STAT3) pathway was determined by Western blot of phosphorylated STAT3 (pSTAT3). A neuron-microglia co-culture system was used to determine whether melatonin can inhibit the neurotoxic effect of pro-inflammatory microglia to post-OGD neurons. RESULTS: Melatonin treatment reduced brain infarct and improved neurological functions 3 days after dMCAO, which was accompanied by decreased expression of pro-inflammatory markers and increased expression of anti-inflammatory markers in the ischemic brain. In vitro studies confirmed that melatonin directly inhibited the pro-inflammatory responses in BV2 cells upon exposure to OGD neuron CM. The microglia possessing pro-inflammatory phenotype exacerbated post-OGD N2a cells death, whereas melatonin reduced such neurotoxic effect. Further, melatonin enhanced the otherwise inhibited pSTAT3 expression in BV2 cells treated with OGD neuron CM. STAT3 blockade significantly reduced the effect of melatonin on microglial phenotype shift. CONCLUSION: Melatonin treatment ameliorates brain damage at least partially through shifting microglia phenotype from pro-inflammatory to anti-inflammatory polarity in a STAT3-dependent manner.