Following ischemic stroke, the progressive formation and mechanical stiffening of the glial scar pose a critical microenvironmental barrier to neural regeneration. However, how this dynamic stiffness influences the fate of neural stem cells (NSCs) remains unclear. To address this, this study aimed to elucidate the mechanotransduction mechanism regulating NSCs differentiation post-stroke. Specifically, thirty male C57BL/6J mice underwent distal middle cerebral artery occlusion to establish a focal cerebral ischemia model. We combined in vivo transplantation of NSCs into distal middle cerebral artery occlusion mouse stroke models and in vitro culture on polyacrylamide hydrogels mimicking scar stiffness. Furthermore, transcriptomic analysis and functional validation through Piezo1 knockdown were employed. Mechanistic insights were explored by examining calcium influx and the Notch signaling pathway. We found that increased substrate stiffness potently inhibited neuronal differentiation and neurite outgrowth. Importantly, Piezo1 was identified as a key mechanosensitive regulator and its knockdown severely diminished both the neuronal differentiated direction and the NSCs proliferation. Mechanistically, Piezo1-mediated calcium influx modulated Notch signaling, forming a stiffness-sensing axis that coordinates the differentiation gene network. Overall, these findings identify aberrant mechanosensing as an important contributing factor of neurogenesis and highlight Piezo1 as a potential therapeutic target for enhancing neural repair.

Background: Reactive astrocytes form a chemical and mechanical glial scar that inhibits neuro-regeneration after stroke. Astrocyte heterogeneity is accompanied by changes in morphology and mechanical properties altering during scar formation after injury. This work aimed to elucidate the relationship between glial scar stiffness and astrocyte subtype transformation. Methods: Astrocyte-specific archaerhodopsin-3 and channelrhodopsin-2 knock-in C57BL/6J mice underwent distal MCAO. Atomic force microscopy, ultrasound elastography and synchrotron radiation were used to determine changes in glial scar stiffness. A proteomic analysis of astrocyte subtypes was performed ex vitro using single-cell laser capture microdissection-MS. Furthermore, optogenetics was employed in vivo to reduce the glial scar stiffness, thereby facilitating neural regeneration following brain injury. Results: Glial scar stiffness systematically increases following stroke and correlates with an increased number of Wnt7b+ fibrotic astrocytes. Furthermore, these results indicate that Piezo1 is the key regulator of astrocytic stiffness and anisotropy, which contributes to the glial scar stiffness in the peri-infarct area. The downregulation of Piezo1 expression promotes activation of the Wnt7b-Ca2+ nonclassical signaling pathway to modulate cytoskeletal reorganization. Finally, the specific optogenetic inhibition of Ca2+ signaling in astrocytes can effectively reduce glial scar stiffness by decreasing the proportion of Wn7b+ astrocytes, which further promotes neuro-regeneration and improves the recovery of motor function after ischemic stroke. Conclusions: This study successfully revealed astrocyte subtype transformation as a key determinant of glial scar physical barrier formation after stroke and highlighted Piezo1 as a potential therapeutic target for modulating the mechanical microenvironment post-injury.

Microglia-mediated neuroinflammation is a key contributor to secondary brain injury across diverse cerebrovascular pathologies, yet the precise transcriptomic diversity and temporal evolution of microglial states across different stroke subtypes remain to be fully elucidated. We utilized single-cell RNA sequencing to profile the temporal transcriptomic landscape of the immune microenvironment in mouse models of ischemic stroke (IS), hemorrhagic stroke (HS), and chronic cerebral hypoperfusion (CCH) at 3, 7 and 14 days post-injury. Our data showed a "temporal mismatch" in immunological trajectories between acute and chronic insults. Specifically, IS and HS triggered an immediate, burst-like microglial activation as early as 3 days, the CCH model exhibited a significant latency window, maintaining a predominantly homeostatic profile until a profound transcriptomic transition toward active phenotypes occurred at 14 days. Notably, we identified a prominent S100A8+/CHIL3+ inflammatory cluster enriched during the hyperacute phase of HS, which may represent an early inflammatory signature associated with intraparenchymal hemorrhage. Interestingly, despite these early divergent responses, the 14-day microglial landscapes across the three models showed a putative shared chronic activation pattern characterized by SPP1+ DAM-like signatures and MHC-II-associated antigenpresentation features. Furthermore, we identified a pre-existing stress-responsive signature (Hspa1a+/ Hspa1b+) in steady-state white matter microglia. These findings provide a high-resolution roadmap of the divergent and convergent immune signatures across cerebrovascular disease spectra, offering hypothesis-generating insights for the development of stage-specific and etiology-tailored immunotherapeutic interventions. Overall, our pooled-library scRNA-seq analysis provides a descriptive and hypothesis-generating framework for strokeassociated microglial and supported by selected tissue-level validation.

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.

OBJECTIVE:Intracranial aneurysm (IA) is a leading cause of subarachnoid hemorrhage, characterized by complex pathogenesis and high mortality rates due to rupture. The aim of this study was to develop a targeted glucagon-like peptide-1 (GLP-1) nanodelivery system to mobilize endothelial progenitor cells (EPCs) and enhance re-endothelialization in a rat model of coiled IA. METHODS:In this study, a matrix metalloproteinase-2 (MMP-2)-targeted nanodelivery platform (hereafter GLP-1@tMSN [targeted mesoporous silica nanoparticle]) based on MSNs functionalized with GLP-1 was developed to mobilize EPCs and accelerate vascular repair. The efficacy of GLP-1@tMSN in promoting EPC recruitment and re-endothelialization was evaluated in a rat coiled aneurysm model, alongside mechanistic studies of the Wnt/β-catenin signaling pathway. RESULTS:In a rat model of coiled IA, GLP-1@tMSN significantly enhanced the recruitment of EPCs and promoted re-endothelialization. Histological analysis demonstrated the formation of mature endothelial-like tissue after 28 days, in contrast to the fibrous tissue observed in the control group. Immunofluorescence analysis confirmed the preferential accumulation of CD34+VEGFR2+ EPCs at the lesion site, with concurrent activation of the Wnt/β-catenin pathway, implicating its pivotal role in driving vascular repair. Preliminary safety evaluations further indicated a favorable biocompatibility profile for the nanotherapeutic system. CONCLUSIONS:The developed functionalized nanodelivery platform represents a promising therapeutic strategy to enhance localized GLP-1 efficacy, facilitating rapid re-endothelialization and potentially reducing long-term recurrence of IAs after embolization. This approach shows substantial potential for improving outcomes for patients with IA.

The formation of glial scar at the chronic stage of neurological disease is mainly attributed to the activation and proliferation of astrocytes, termed astrogliosis. It is documented that astrogliosis-induced glial scar formation following stroke severely impairs neurological recovery. However, the mechanisms underlying astrogliosis remain poorly understood. Herein, we discovered that knockdown of MEGF10, a well-known phagocytic receptor that mediates astrocyte-dependent synapse engulfment, caused G1 phase arrest in astrocytes and downregulated cell cycle-related genes, leading to reduced astrocyte proliferation and activation. Genetic deletion of MEGF10 in astrocytes reduced astrocyte proliferation and activation, glial scar formation, and extracellular matrix deposition, subsequentially decreased brain atrophy and promoted neurofunction recovery of mice after stroke. Leveraging on these findings, we further developed a clinically applicable lipid nanoparticle (LNP) system with good biocompatibility, capable of targeted delivery of MEGF10 siRNA to astrocytes. Injecting these LNPs effectively reduced astrocyte proliferation and activation, minimized glial scar formation, and enhanced neurobehavioral recovery of mice after stroke. Our study unveils a previously unrecognized role of MEGF10 in regulating astrogliosis and provides a clinically translatable strategy for targeted modulating glial scar formation and promoting neurofunction recovery after stroke, suggesting that targeting MEGF10 may represent a new therapeutic approach for treating stroke.

The vascular hyperinflammatory microenvironment significantly influences the regeneration and repair of central nervous system (CNS) following injury. Training immune behavior of immune cells under stress conditions is crucial for maintaining vascular microenvironment homeostasis and restoring vascular function. In this study, we engineer a novel hyperinflammatory regulatory peptide (mND13) by rational grafting of a DJ-1-derived peptide with an MMP-2 responsive peptide motif. This modified peptide is further conjugated onto hyaluronic acid methacrylate (HAMA) microspheres via photo-click chemistry and microfluidic technology, generating mND13@HAMA microspheres. This platform induces microglia immuno-training, inhibits their pro-inflammatory polarization, thus orchestrating neurovascular niche remodeling and promoting neural recovery through immune-vascular crosstalk. Both in vitro and in vivo investigations show that these immune-functionalized peptide microspheres mND13@HAMA drastically decrease endothelial cell apoptosis and the expression of pro-inflammatory molecules like iNOS, CD86 and IL-1 beta in microglia. To further enhance post-stroke angiogenesis, we co-deliver VEGF liposomes with mND13@HAMA, which is proven to promote vascular sprouting and growth while inhibiting vascular apoptosis. This combined system markedly reduces brain atrophy volume in stroke mouse, improves neurobehavioral functions, and enhances angiogenesis. In conclusion, this immunomodulatory microsphere, capable of training immune cell behavior, holds significant therapeutic value for treating stroke along with other CNS injuries.

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.

BACKGROUND:Ischemic stroke remains a leading cause of long-term disability and mortality worldwide, with few effective treatment options. A key challenge in recovery is the brain's limited capacity to regenerate neurovascular structures after injury. To address this, we developed a dual-functionalized extracellular vesicle (EV) platform designed to enhance both targeting specificity and therapeutic efficacy for post-stroke repair. METHODS:Neural stem cell-derived EVs were bioengineered via bio-click chemistry to display RGD peptides, enabling selective binding to integrin αVβ3, which is upregulated on activated endothelial cells in ischemic regions. EVs were concurrently loaded with vascular endothelial growth factor (VEGF), a pro-angiogenic and neurogenic cytokine that also enhances αVβ3 expression-thus creating a synergistic positive feedback mechanism to amplify targeting and tissue repair. RESULTS:Engineered EVs retained normal morphology and showed a 5.2-fold increase in endothelial uptake compared to naïve EVs (p < 0.01). In vitro, they significantly enhanced endothelial cell migration by 2.1-fold (p < 0.05). In a mouse model of transient middle cerebral artery occlusion (tMCAO), intravenously delivered dual-functionalized EVs preferentially accumulated in the ischemic hemisphere, reduced infarct volume by 52.4%, and improved motor coordination (rotarod latency) by 71.8% compared to PBS-treated controls (p < 0.05). Immunostaining revealed enhanced CD31+ microvessel density and increased Nestin+ neural stem and progenitor cell presence, indicating promotion of both angiogenesis and neurogenesis. CONCLUSION:This study presents a dual-functionalized EV system that combines targeted delivery with therapeutic reinforcement through VEGF loading, offering a potent and synergistic approach for ischemic stroke repair. These findings support further translational development of engineered EVs for neurovascular regeneration.

Extracellular vesicles (EVs) have shown great potential for treating various diseases. Translating EVs-based therapy from bench to bedside remains challenging due to inefficient delivery of EVs to the injured area and lack of techniques to visualize the entire targeting process. Here we developed a dopamine surface functionalization platform that facilitates easy and simultaneous conjugation of targeting peptide and multi-mode imaging probes to the surface of EVs. Utilizing this platform we concurrently modified M2 microglia-derived EVs (M2-EVs) with neuronal targeting peptide rabies virus glycoprotein peptide 29 (RVG29) and multi-modal imaging tracers, resulting in the targeted delivery of M2-EVs to stroke mice brain and enabled the dynamic visualization of the targeting process from whole-body to cellular levels. We determined that intra-arterial injection achieved the highest efficiency of targeted delivery of engineered EVs to the stroke mice brain, improved therapeutic efficacy by reducing neuronal apoptosis. Mechanistically, EVs miRNA array revealed that a number of anti-apoptosis related miRNAs were significantly up-regulated, including miR-221-3p and miR-423-3p, both exerted anti-apoptotic effects through p38/ERK signalling pathways in stroke. Overall, this platform provides a facile and powerful tool for multifunctional engineering of EVs for multiscale therapeutic evaluation and enhancement of EV-based therapy, with valuable prospects for clinical translation.

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.

Stem cell therapy for stroke is hindered by poor cell survival and integration within the ischemic microenvironment. Injectable microgels have been widely employed in cell delivery for their ability to promote cell infiltration, but dense hydrogel networks often limit cell survival. Here we present an injectable microgel-matrix composite scaffold that combines microporous microgels for neural progenitor cells (NPCs) delivery with interstitial spaces to support vascular growth. Using a gas-shearing fabrication approach, primary rat NPCs are efficiently encapsulated in phase-separated microporous microgel (PSMM), exhibiting superior survival and proliferation. In vitro studies demonstrate that PSMM scaffolds support endothelial cell (EC) sprouting and vascular formation, and in ischemic stroke rats, this formulation significantly enhance NPC loading capacity, survival, and differentiation, along with increased EC proliferation and infiltration. Ultimately, the microgel-matrix scaffold enhances long-term neurological recovery in stroke models, offering an efficient strategy that couples high cell loading with vascularization to advance regenerative medicine.

Humans are widely exposed to phthalates, a common chemical plasticizer. Previous cohort studies have revealed that maternal exposure to monobutyl phthalate (MBP), a key metabolite of phthalates, is associated with neurodevelopmental defects. However, the molecular mechanism remains unclear. Here, we demonstrate that maternal exposure to MBP enhances neural stem cell (NSC) differentiation into astrocytes with highly expressed C3 and LCN2 in mouse offspring, resulting in increased synapse phagocytosis and cognitive dysfunction. Mechanistically, we find that MBP exposure activates the IRE1α/XBP1s (spliced XBP1) stress response pathway, which regulates key genes involved in astrocyte differentiation (SOX9 and ATF3) and reactivity (C3 and LCN2). Conditional knockout or pharmacological inhibition of IRE1α markedly inhibits NSC differentiation into astrocytes and astrocyte reactivity, attenuates synapse phagocytosis, and improves cognitive function. This phenotype is further recapitulated in a human brain organoid model. Together, these findings unveil the molecular mechanism underlying the neurodevelopmental deficits caused by a widespread environmental pollutant.

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.