Yaohui Tang

Aims: To investigate the role of the RIPK1 inhibitor GFH312 in ischemic stroke and identify its potential mechanisms. Methods: GFH312 was administered to adult mice in an ischemic stroke model. To ascertain the effectiveness of treatment, neurobehavioral tests and assessments of brain damage were carried out. Brain tissues were examined using immunofluorescence as well as western blot and real‐time quantitative PCR at various intervals following ischemia to evaluate inflammatory gene expression and signaling pathways linked to apoptosis and necroptosis. Results: Temporal analysis revealed that necroptosis and apoptosis were rapidly activated following ischemia, peaking at 1–3 days. GFH312 significantly improved neurological function and reduced brain injury in stroke mice. Mechanistically, GFH312 markedly inhibited apoptosis and necroptosis via suppression of the RIPK1‐related signaling pathway (RIPK1‐RIPK3‐MLKL). In addition, GFH312 greatly decreased inflammatory gene expression in the brain. Conclusion: Apoptosis and necrotic apoptosis occur primarily during the acute poststroke phase. GFH312 exerts neuroprotective effects after ischemic stroke, which are associated with suppression of RIPK1‐related apoptosis, necroptosis, and neuroinflammation.

Background: Tensor‐valued diffusion magnetic resonance imaging enables the separation of total kurtosis into isotropic and anisotropic components, offering improved specificity over conventional diffusion kurtosis imaging. In this study, we introduce a novel framework for detecting anisotropic‐isotropic kurtosis mismatch and evaluate its relevance in experimental ischemic stroke. Methods: Tensor‐valued diffusion magnetic resonance imaging was performed in a rat model of middle cerebral artery occlusion. Metrics, including anisotropic mean kurtosis and isotropic mean kurtosis, were quantified to assess microstructural tissue heterogeneity. Histologic validation was conducted using coregistered tissue sections. Results: We observed significant mismatches between mean diffusivity, mean kurtosis, as well as specifically in anisotropic mean kurtosis and isotropic mean kurtosis in ischemic regions. Notably, anisotropic mean kurtosis and isotropic mean kurtosis exhibited statistically significant alterations compared with contralateral regions. Moreover, these metrics strongly correlated with pathologic findings. The observed mismatch highlighted microstructurally distinct subregions within the lesion. Conclusions: This study establishes an in vivo framework for characterizing anisotropic‐isotropic kurtosis mismatch in an experimental stroke model. By quantifying the isotropic mean kurtosis/anisotropic mean kurtosis mismatch, tensor‐valued diffusion magnetic resonance imaging provides a more specific mechanistic characterization of diffusion heterogeneity than conventional diffusion tensor imaging and diffusion kurtosis imaging metrics, supporting its use as a preclinical tool for investigating microstructural alterations in ischemic brain tissue.

Promoting white matter repair is crucial for functional recovery after ischemic stroke. Our previous study has identified an IFN‐ γ ‐responsive microglia phenotype in the brain after stroke. However, its role in white matter repair remains poorly understood. Herein, we find that extracellular vesicles derived from IFN‐ γ ‐responsive microglia (IFN‐ γ EVs) are internalized by oligodendrocyte precursor cells (OPCs), leading to impaired OPCs proliferation, survival, and differentiation and reduced white matter repair in a murine transient middle cerebral artery occlusion (tMCAO) model. Mechanistically, miR‐9‐5p enrichment in IFN‐ γ EVs suppresses NEDD4 expression, a key regulator of myelination, leading to reduced OPCs maturation and white matter repair. Genetic interventions including miR‐9‐5p knockdown in IFN‐ γ EVs or NEDD4 overexpression in OPCs restore remyelination and functional recovery. Our findings demonstrate that IFN‐ γ EVs act as negative regulators of OPCs differentiation and remyelination following ischemic stroke via miR‐9‐5p/NEDD4 pathway. Targeting this signaling axis may represent a novel therapeutic strategy to enhance remyelination and functional recovery after stroke.

Background: Previous studies have reported the presence of interferon‐responsive microglia in the brain after central nervous system injury. However, their roles and the underlying mechanisms in neurological function recovery remain poorly understood. Methods: Adult male mice were subjected to 90‐minute transient middle cerebral artery occlusion, and brain tissues were analyzed using single‐cell RNA sequencing (scRNA‐seq) at 14 days after stroke. Immunostaining, quantitative real‐time polymerase chain reaction and ELISA were conducted to validate the presence of interferon‐γ–responsive microglia in stroke mice brains. Extracellular vesicles (EVs) were isolated from interferon‐γ–treated BV2 microglia via ultracentrifugation. Interferon‐γ EVs were then used to treat neural stem cells (NSCs) in vitro or administered intravenously to mice every other day, starting at 7 days after transient middle cerebral artery occlusion. Neurobehavioral tests, cresyl violet staining, Golgi staining, and immunostaining were performed to evaluate NSC differentiation, neurogenesis, and neurobehavioral recovery. Micro RNA (miR) sequencing and bioinformatic analysis were conducted to explore targeted genes and signaling pathways underlying interferon‐γ EV‐mediated inhibition of neurogenesis. Results: Single‐cell RNA sequencing, immunostaining, quantitative real‐time polymerase chain reaction, and ELISA showed the presence of interferon‐γ–responsive microglia in stroke mice brains. Interferon‐γ EVs were internalized by NSCs, leading to reduced NSC survival and neuronal differentiation. Administration of interferon‐γ EVs increased brain atrophy volume, inhibited neurobehavioral recovery and neurogenesis in mice after stroke. miRNA array revealed 12 upregulated microRNAs, and treatment with miR‐199a‐5p mimic inhibited the survival and neuronal differentiation of NSCs, and knockdown of miR‐199a‐5p in interferon‐γ EVs increased neurogenesis in stroke mice. miRNA database analysis and luciferase reporter assay identified SIRT1 as a downstream target gene of miR‐199a‐5p. Treatment with SIRT1 agonist promoted the survival and neuronal differentiation of NSCs, confirming that interferon‐γ EVs inhibited neurogenesis via miR‐199a‐5p/ SIRT1 . Conclusions: Our study demonstrated that interferon‐γ EVs inhibited the survival and neuronal differentiation of NSCs, exacerbating brain injury via the miR‐199a‐5p/ SIRT1 axis after ischemic stroke, providing a novel target for treating ischemic stroke.

Astrocyte-mediated synaptic phagocytosis plays a critical role in regulating brain development and disease progression. Our previous studies have demonstrated that MEGF10, a key molecule mediating astrocytic phagocytosis of synapses, is remarkably upregulated during the chronic phase of stroke, leading to synapse loss and exacerbating brain injury. Targeting MEGF10 to reduce synapse engulfment has emerged as a promising therapeutic strategy for stroke. However, achieving precise astrocyte-targeted modulation remains a significant challenge, primarily due to the absence of efficient and specific gene delivery systems. Lipid nanoparticles (LNP) functionalized with peptides or antibodies are promising gene delivery vectors for treating central nervous system (CNS) diseases. Herein, we developed an LNP system loaded with MEGF10 siRNA and functionalized with an astrocyte-targeting peptide AS1 (siMEGF10-LNP@AS1). Ex vivo imaging and immunostaining results showed that injection of siMEGF10-LNP@AS1 efficiently delivered MEGF10 siRNA to astrocytes, downregulated MEGF10 expression in stroke mice, and reduced astrocytic phagocytosis of synapses. This intervention enhanced synaptic density while maintaining good biocompatibility. Furthermore, administration of siMEGF10-LNP@AS1 improved motor and cognitive function and reduced brain atrophy volume in stroke mice. Overall, our study establishes a robust platform for astrocyte-targeted gene modulation, offering a potential therapeutic strategy for CNS diseases.

Astrocyte activation and gender differences play critical roles in the prognosis following stroke. Recent studies have shown that optogenetic technology can promote brain repair after stroke by activating astrocytes in male rats. However, it remains unclear whether gender differences influence the efficacy of optogenetic activation of astrocytes in regulating post-stroke brain repair and its underlying mechanisms. In this study, we activated astrocytes in the ipsilateral cortex of adult glial fibrillary acidic protein-channelrhodopsin 2-enhanced yellow fluorescent protein (GFAP-ChR2-EYFP) transgenic Sprague Dawley rats using optogenetic stimulation at 24, 36, 48, and 60 h after inducing photothrombosis stroke. Neurobehavioral tests, cresyl violet staining, RT-qPCR, Western blot, and immunofluorescence analysis were performed on both female and male rats. Our results showed that male rats exhibited significant improvements in behavioral scores and reduction in infarct size after optogenetic activation of astrocytes at three days post-stroke (p < 0.05), whereas no significant changes were observed in female rats. Additionally, in female rats, the expression of basic fibroblast growth factor (bFGF) increased after ischemic stroke and astrocytic optogenetic stimulation (p < 0.05), leading to enhanced endothelial cell proliferation compared to male rats (p < 0.05). In vitro experiments further demonstrated that the astrocyte activation was inhibited in the presence of bFGF (p < 0.05). These findings suggest that the increase in bFGF levels in females following stroke may inhibit the optogenetic activation of astrocytes, thereby attenuating the therapeutic effect of astrocyte activation on post-stroke brain repair. This study provides important insights into the gender-specific roles of astrocytes in the acute phase of ischemic stroke.

Background: Tensor-valued diffusion MRI (dMRI) enables the separation of total kurtosis into isotropic and anisotropic components, offering improved specificity over conventional Diffusion Kurtosis Imaging (DKI). In this study, we introduce a novel framework for detecting anisotropic-isotropic kurtosis mismatch and evaluate its utility in characterizing stroke lesions. Methods: We performed tensor-valued dMRI in a rat model of middle cerebral artery occlusion (MCAO). Metrics including anisotropic kurtosis (MK _A ) and isotropic kurtosis (MK _I ) were quantified to assess microstructural tissue heterogeneity. Histological validation was conducted using co-registered tissue sections. Results: We observed significant mismatches between mean diffusivity (MD), mean kurtosis (MK), as well as specifically in MK _A and MK _I in ischemic regions. Notably, MK _A and MK _I exhibited statistically significant alterations compared to contralateral regions. Moreover, these metrics strongly correlated with pathological findings. The mismatch highlighted microstructurally distinct subregions within the lesion. Conclusions: This is the first study to propose an anisotropic-isotropic kurtosis mismatch framework in vivo. Tensor-valued dMRI may offer a more specific imaging biomarker for stroke, with potential for improving lesion subtyping and guiding therapeutic decisions.

Due to the limited effects of current treatments on brain repair and regeneration, stroke continues to be the predominant cause of death and long-term disability on a global scale. In recent years, hydrogel-based biomaterials combined with stem cells and extracellular vesicles have emerged as promising new treatments to improve brain regeneration after stroke. However, the clinical translation of hydrogel-based biomaterials for the treatment of brain injury is still far from satisfactory. In this review, we first summarise the present status of stroke-related clinical treatments and the advantages provided by hydrogel-based materials in combination with stem cells and extracellular vesicles in preclinical studies. We then focus on the possible causes of the gap between preclinical studies and clinical translation of hydrogel-based biomaterials from the perspective of biocompatibility and safety, the choices of preclinical models, the lack of clinical noninvasive imaging methods, standardisation and quality control, manufacturing scalability, and regulatory compliance. With the progress in the abovementioned areas, we believe that the clinical translation of hydrogel-based biomaterials will greatly improve brain regeneration after stroke and that this improvement will be realised by the general public in the near future.

Epilepsy, a neurological disorder marked by recurrent, unprovoked seizures, is often linked to dysregulated iron metabolism, resulting in iron overload and subsequent cellular dysfunction or death within epileptogenic regions. We proposed a specific, noninvasive technique using paramagnetic susceptibility imaging via magnetic resonance imaging to quantify in vivo brain iron levels, aiming to enhance our understanding of epilepsy pathology and improve diagnostic accuracy. Our imaging and histopathological studies demonstrated that paramagnetic susceptibility is a sensitive biomarker for iron quantification in epilepsy. This method effectively detects iron abnormality from various causes and highlights that iron alters within epileptogenic zones, indicating the presence of potentially salvageable tissue. Furthermore, iron accumulation was observed to disrupt cortical laminar structures in epileptogenic zones and was associated with the proliferation of central nervous system cells, particularly astrocytes. Paramagnetic susceptibility imaging provides previously unknown insights into epilepsy, offering potential applications in diagnostics, monitoring, and personalized treatment strategies.

The crosstalk between immune cells and the neurovascular unit plays a pivotal role in neural regeneration following central nervous system (CNS) injury. Maintaining brain immune homeostasis is crucial for restoring neurovascular function. In this study, an interactive bridge was developed via an immunomodulatory hydrogel microsphere to link the interaction network between microglia and the neurovascular unit, thereby precisely regulating immune-neurovascular crosstalk and achieving neural function recovery. This immunomodulatory crosstalk microsphere (MP/RIL4) was composed of microglia-targeted RAP12 peptide-modified interleukin-4 (IL-4) nanoparticles and boronic ester-functionalized hydrogel using biotin-avidin reaction and air-microfluidic techniques. We confirmed that the immunomodulatory microspheres reduced the expression of pro-inflammatory factors including IL-1β, iNOS, and CD86, while upregulating levels of anti-inflammatory factors such as IL-10, Arg-1, and CD206 in microglia. In addition, injection of the MP/RIL4 significantly mitigated brain atrophy volume in a mouse model of ischemic stroke, promoted neurobehavioral recovery, and enhanced the crosstalk between immune cells and the neurovascular unit, thus increasing angiogenesis and neurogenesis of stroke mice. In summary, the immunomodulatory microspheres, capable of orchestrating the interaction between immune cells and neurovascular unit, hold considerable therapeutic potential for ischemic stroke and other CNS diseases.