Hydroxyl radical (center dot OH) is the most reactive and deleterious reactive oxygen species, yet its direct detection remains highly challenging because of its extremely short lifetime and high reactivity. Here we report the HKOH-2 series of fluorescent probes for imaging center dot OH in living cells, rat brain slices, and living zebrafish. In addition, HKOH-2L and HKOH-2m were developed for selective detection of center dot OH with organelle resolution in lysosomes and mitochondria, respectively. The HKOH-2 series thus provides versatile chemical tools for monitoring local center dot OH production in living cells and in vivo.

Background: Post-stroke brain stimulation is a promising neurorestorative strategy, yet the underlying molecular mechanisms driving recovery remain unclear. Our prior work demonstrated that post-stroke optogenetic stimulation of the ipsilesional motor cortex (iM1) enhances functional recovery, and our RNA sequencing suggested cholesterol metabolism as a key pathway modulated by stimulation. Here, we examined the temporal dynamics of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), a key cholesterol enzyme, after stroke and assessed how optogenetic iM1 stimulation influences this expression. Methods: Male C57BL/6 mice (6-7 weeks) underwent stereotaxic surgery to express Channelrhodopsin in iM1 excitatory neurons and optical fiber implantation. After 5-6 weeks, mice received transient middle cerebral artery occlusion (30 min). Optogenetic stimulations were delivered from post-stroke days (PD) 5–14. Motor performance was assessed using the rotating beam test at pre-stroke baseline, PD4, 7 and 14. Brains were collected from stroke (PD1, 7&15) and sham control mice (n=4-5/group), and processed for immunohistochemistry using antibodies against HMGCS1, NeuN and CD68. Results: iM1 stimulation enhanced recovery at PD14, improving beam performance in both travel distance and speed (p<0.05). Previous RNA sequencing revealed involvement of multiple cholesterol biosynthesis and metabolism pathways at PD15 in iM1-stimulated mice. Immunostaining demonstrated that HMGCS1 was primarily expressed in primary motor cortex neurons and in peri-infarct glia. Compared with sham, iM1 showed a transient HMGCS1 increase at PD1, followed by reduced levels at PD7 and PD14, while contralateral M1 displayed reduced expression at all time points. At PD15, neuronal HMGCS1 in both stimulated and non-stimulated iM1 was significantly lower than in their respective contralateral M1 (p<0.05). In both regions, stimulated mice showed a trend toward higher HMGCS1 than non-stimulated mice. Stimulated mice also showed increased glial HMGCS1 in the peri-infarct region. Conclusions: Our findings link optogenetic stimulation-induced recovery to modulation of cholesterol metabolism, highlighting HMGCS1 as a potential molecular contributor. Future studies will validate cholesterol-related gene changes and clarify cell-type-specific contributions, with implications for targeting metabolic pathways to enhance neurorestoration.

Introduction: Acute hyperglycemia affects ~40% of stroke patients and worsens outcomes despite standard glucose control, yet no targeted therapy exists. We identify a previously unrecognized Metabolic–Complement–Vascular (MCV) axis, where hyperglycemia rapidly disrupts the endothelial glycocalyx and activates vascular complement within hours of stroke onset, defining a new, time-sensitive therapeutic target. Method: Male C57BL/6 mice (10-11 weeks) underwent 30 min transient MCAO with reperfusion to mimic thrombectomy. Hyperglycemia was induced by intraperitoneal glucose injection 10 min before occlusion. BBB disruption was assessed at multiple time points by Evans blue or IgM/IgG staining; neurological deficits, motor function (open field), and mortality were recorded. Glycocalyx disruption was measured by electron microscopy and IB4 staining; complement activation assessed by C3d immunostaining. The role of complement C3 was tested using C3 knockout mice and targeted inhibitor CR2-Crry. Human post-mortem ischemic stroke and control brain tissues (n = 5/group) were analyzed for glycocalyx integrity (UEA I lectin) and vascular immune injury (C3d/IgG). Pre-thrombectomy plasma complement markers (n=66) were analyzed via elastic net regression to predict outcomes. Result: Hyperglycemia caused rapid and severe luminal vascular injury within 4.5 h of stroke, with glycocalyx loss, luminal IgM/IgG deposition, vascular C3 activation, and BBB leakage (n=5, p<0.0001); not observed in normoglycemic stroke. Complement activation persisted after glucose normalization, was exacerbated by reperfusion, and propagated into the brain. This early vascular damage increased mortality (100% vs. 25%, p=0.0008) and worsened neurological deficits (p<0.001). C3 knockout mice had reduced BBB leakage (n=6, p<0.01) and improved function (p<0.01). Targeted C3 inhibition with CR2-Crry at 30 min post-reperfusion preserved BBB integrity and improved function (n=4, p<0.01), providing proof-of-concept for adjunct complement-targeted therapy. In human stroke brain, C3 activation colocalized with luminal glycocalyx loss. In human pre-thrombectomy plasma circulating complement activation markers (Ba, Bb, C4a, C3a) independently predicted modified Rankin Scale outcomes at discharge. Conclusion: These findings reframe acute hyperglycemic stroke as a rapid luminal vascular disorder and identify complement inhibition as a promising adjunct to reperfusion therapy.

Introduction: The cross-talk between the brain and peripheral immune organs plays a crucial role in the response to stroke injury. Spleen responses are implicated in stroke pathology and inflammatory responses. Hyperglycemia is known to worsen stroke outcomes, with increased immune cell infiltration into the brain. However, it remains unclear whether hyperglycemia exacerbates spleen immune cell responses and whether this contributes to the heightened immune response in hyperglycemic stroke. Method: Male C57/BL6 mice (10-12 weeks) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 minutes, followed by reperfusion to mimic ischemic stroke. Acute hyperglycemia was induced by glucose injection 10 minutes before MCAO. The study included three groups: sham, MCAO only, and MCAO with hyperglycemia (n=6-7 per group). At 24 hours post-stroke, spleen and brain immune cell populations were analyzed using our established 12-color flow cytometry technique, which simultaneously analyzes myeloid and lymphoid subpopulations. Results: Compared with sham controls, stroke significantly decreased the numbers of dendritic cells (2.33 ± 0.48 vs. 1.50 ± 0.42 million; p<0.05), NK cells (2.30 ± 0.37 vs. 1.21 ± 0.37 million; p<0.01), Ly6C+ macrophages (0.29 ± 0.12 vs. 0.11 ± 0.06 million; p<0.01), and total lymphocytes (56.9 ± 16.6 vs. 37.0 ± 5.1 million; p<0.05) at 24 hours post-stroke in the spleen, whereas there were no significant changes in neutrophils and Ly6C+ monocytes cell counts. Hyperglycemia during stroke did not alter spleen immune cell numbers compared to stroke alone. However, hyperglycemia significantly increased brain-infiltrating immune cells at 24 hours after stroke, including neutrophils, B cells, and CD8+ T cells compared to normoglycemic mice. Conclusion: This study provides insights into spleen immune cell responses to acute ischemic stroke and hyperglycemia. Significant changes in spleen immune cell populations after stroke confirm their potential roles in stroke pathology and recovery, warranting further investigation. Acute hyperglycemia, however, does not significantly affect spleen immune cell numbers after stroke, implying that the spleen is not the primary target of hyperglycemia-exacerbated immune cell infiltration into ischemic stroke brains. These results pave the way for exploring other mechanisms driving immune cell infiltration under hyperglycemic conditions.

Introduction: Post-stroke brain stimulation is a promising neurorestorative approach, yet the molecular mechanisms driving recovery remain unclear. Our previous work demonstrated that post-stroke optogenetic stimulations of the ipsilesional primary motor cortex (iM1) promotes functional recovery. To understand the mechanisms driving post-stroke recovery, we investigated the transcriptome of iM1 in non-stimulated and stimulated mice using RNA sequencing. Methods: C57Bl6 male mice underwent stereotaxic surgery to express Channelrhodopsin in iM1 excitatory neurons, with optical fiber implanted in the same location. After 5-6 weeks, mice underwent transient middle cerebral artery occlusion (30 minutes). Stimulated mice received optogenetic stimulations from post-stroke days (PD) 5–14. Rotating beam test was performed at pre-stroke baseline, PD4, 7 and 14. iM1 from stimulated, non-stimulated stroke mice at PD 7&15, and sham mice were processed for RNA sequencing (n=4-5/group). Expression of cholesterol enzymes such as HMGCS1 was examined using quantitative PCR and immunohistochemistry. Cholesterol levels were visualized using filipin or BODIPY. Results: iM1 stimulations enhanced recovery at PD14, with longer distance traveled and faster speed on the rotating beam test (p<0.05). RNA sequencing revealed a distinct transcriptomic landscape between stimulated and non-stimulated stroke mice. In non-stimulated mice, 4526 differentially expressed genes (DEGs) were identified in iM1 at PD7, and number of DEGs dropped to 95 by PD15, compared to sham controls. In contrast, stimulated mice exhibited 1446 DEGs at PD7, which remained elevated at 1090 DEGs by PD15. Ingenuity pathway analysis highlighted downregulation of multiple cholesterol biosynthesis and metabolism pathways in iM1-stimulated mice. qPCR confirmed that iM1 stimulation reduced the expression of cholesterol enzyme HMGCS1 in iM1. Preliminary BODIPY and HMGCS1 staining data indicated noticeable cholesterol level changes in iM1 post-stroke. Conclusions: Our transcriptome data revealed important insights into the molecular signaling of optogenetic stimulation-induced recovery, particularly cholesterol metabolism. Ongoing studies include immunostaining of cholesterol metabolism related genes and co-staining of cholesterol levels with cell type specific markers (neurons, glia, microglia). These data suggest that reduction in cholesterol accumulation may be a key mechanism in post-stroke recovery.

Background: A damaged stroke area can affect both local and connected brain regions, leading to network-wide disruptions in brain functions. Increasing evidence shows that estrogen, particularly 17β-estradiol, plays a protective role after ischemic stroke. Understanding how hormonal levels impact brain stimulation may shed light on varying responses to neuromodulation therapies across estrous cycle phases. Previously, we demonstrated that optogenetic stimulations in the ipsilesional primary motor cortex (iM1) enhance functional recovery in male mice. In this study, we aim to expand upon these findings by exploring functional recovery and post-stroke neuroplasticity changes in in female mice. Methods: Female C57BL/6 mice (7-9 weeks old) underwent stereotaxic surgery to express channelrhodopsins-2 and implant an optical fiber in iM1. Four weeks later, transient middle cerebral artery occlusion (MCAO) was induced. Optogenetic stimulation was initiated on day 5 post-stroke and continued for 10 days. Estrous cycles were monitored via vaginal swabbing and estradiol ELISA measurements. Rotating beam tests were performed at pre-stroke, 4, 7, 10, and 14 days post-stroke (PD4, PD7, PD10, and PD14). Infarct size, astrocytic activation and microglia/macrophage activation were assessed. Results: Rotating beam tests showed that 71.4% of the stimulated mice significantly recovered by PD10 (p<0.01). We observed irregular hormonal cycles post-stroke, with the diestrus phase noticeably longer in majority of mice. Stimulated mice exhibit a trend toward better recovery when in the diestrus/metestrus phase at the start of stimulation on PD5. Optogenetic stimulation appeared to reset the hormonal cycles, though some irregularities remained during stimulation period. Additionally, recovery varied during estrous cycle stages, with optogenetic stimulation enhancing motor function during specific phases. At PD10, improved recovery was significantly correlated with low estradiol levels (R 2 = 0.61, p<0.05), and most recovered mice were in the estrus phase by PD14. Conclusions: These results highlight the importance of considering hormonal cycles in post-stroke brain stimulations. Ongoing studies are investigating the effects of stimulation on hormonal cycles in relation to post-stroke plasticity. Our data suggest that tailoring brain stimulations to specific estrous hormonal phases may optimize recovery outcomes.

Background:Acute hyperglycemia affects approximately 40% of stroke patients and is associated with worse outcomes. The underlying mechanisms linking this metabolic stress to stroke-induced brain injury remains unclear, and effective therapies are lacking. Methods:In a mouse model of acute hyperglycemic stroke, luminal disruption, blood-brain barrier (BBB) leakage, neurological deficit, motor function, and mortality were evaluated. Vascular luminal glycocalyx and complement activation were assessed by immunostaining, with glycocalyx loss confirmed by electron microscopy. Complement C3's causal role was tested using C3 knockout mice and site-targeted inhibition with CR2-Crry. To enhance translational relevance, post-mortem human stroke and control brains were immunostained to assess the association between endothelial glycocalyx loss and vascular complement activation. In a separate stroke patient cohort, soluble complement activation products were measured in pre-thrombectomy plasma, and their predictive value for modified Rankin Scale (mRS) outcomes evaluated using elastic net regression. Results:Hyperglycemic stroke mice exhibited accelerated and more severe BBB breakdown, greater functional deficits, and higher mortality than normoglycemic controls, mirroring clinical observations. Acute hyperglycemia triggered rapid vascular luminal injury characterized by loss of endothelial luminal glycocalyx, luminal IgM/IgG deposition, and vascular complement C3 activation, leading to BBB disruption. This vascular luminal injury was corroborated in human stroke brain tissue. These luminal changes persisted despite glucose normalization and were exacerbated by reperfusion, driving injury into the brain parenchyma. Genetic and pharmacological approaches confirmed vascular complement activation as a causal driver of severe BBB disruption and poor outcomes. Importantly, site-targeted pharmacological inhibition of complement after reperfusion preserved BBB integrity and improved outcomes, defining a time-specific, luminal-directed strategy as a promising adjunct to thrombectomy. Notably, soluble complement activation markers in pre-thrombectomy stroke plasma predicted clinical outcomes, highlighting their potential as pre-intervention markers for patient stratification and tailored therapy. Conclusion:This study reframes acute hyperglycemic stroke as a vascular luminal disorder, establishing a novel Metabolic-Complement-Vascular (MCV) axis linking metabolic stress to endothelial luminal glycocalyx loss, vascular complement activation, and BBB breakdown in both mice and humans. This new mechanistic understanding transforms the therapeutic landscape of hyperglycemic stroke, offering a potential time-defined, luminal-focused adjunct therapy alongside thrombectomy. Clinical Perspective:What Is New?: - This study reframes hyperglycemic stroke as an acute vascular luminal problem, marked by rapid loss of endothelial luminal glycocalyx and complement C3 activation at the vascular luminal surface. - The rapid luminal changes, identified in both rodent and human stroke brain tissues, establish a novel Metabolic-Complement-Vascular (MCV) axis linking metabolic stress to luminal damage, blood-brain barrier (BBB) breakdown, and injury progression into the brain parenchyma. - The first clinical evidence that pre-thrombectomy plasma complement activation markers independently predict stroke outcomes - laying the foundation for risk stratification before reperfusion and precision adjunct therapies.What Are the Clinical Implications?: - The rapidity and persistence of the MCV axis activation-even after glucose normalization-help explain the limited efficacy of insulin therapy, shifting the therapeutic focus from glycemic control to luminal-targeted interventions.- Identification of a narrow but actionable window following reperfusion where complement C3 inhibition preserves the BBB and limits injury progression to the parenchyma, offers a promising adjunct to thrombectomy in metabolically vulnerable stroke patients.- Targeting the vascular luminal surface reshapes the therapeutic landscape for hyperglycemic stroke by enabling systemic interventions-bypassing the challenge of BBB penetration, supporting rapid clinical translation using existing FDA-approved C3 inhibitors, and framing the endothelial glycocalyx as a promising area for therapeutic and diagnostic exploration in hyperglycemic stroke and broader cerebrovascular disease.

Introduction: Previous stroke studies in female rodents have demonstrated that the severity of ischemic damage is differentially impacted by the estrous cycle. Estrogen is considered to be neuroprotective, but it is unclear whether this protection is dependent on the vasodilator properties of estrogen. In this study, we investigated the impact of estrus cycle stage on stroke lesion volume and whether that impact was dependent on cerebral blood flow (CBF) changes during stroke. Methods: Ischemia was induced in adult ( 10 weeks) male and female Sprague-Dawley rats by 30 minutes middle cerebral artery occlusion (MCAO) using the suture model. Prior to the stroke procedure, the phases of the estrous cycle (proestrus(P), estrus(E), metestrus(M) and diestrus (D)) were determined by vaginal smear tests in female rats. Cerebral blood flow (CBF) was measured by laser doppler and infarct volume were measured by TTC staining at d2 post-stroke. Results: Females in proestrus and diestrus (P/D) stages of the estrous cycle, which have reportedly high levels of estrogen, had significantly (p<0.05) smaller CBF decreases during ischemia and a trend for smaller infarcts than those in estrous and metestrus stages, which have lower estrogen levels. This suggests that the protective effects of estrogen may be related to effects on blood flow. However, when compared to male rats, females in P/D stages had significantly (p<0.05) smaller brain infarcts despite having similar CBF decreases. This implies that additional mechanisms are responsible for the neuroprotection observed in females when compared to males. Conclusion: Estrous cycle stage affects CBF decrease during ischemia, but this does not account for the difference in infarct size observed between male and female rats. Our study indicates that multiple mechanisms are responsible for ischemic tolerance in specific stages of the estrous cycle. Keywords: stroke, estrous cycle, CBF, estrogen

BACKGROUND:Angong Niuhuang Wan (AGNHW, ), is a classical medicinal formula in Traditional Chinese Medicine (TCM) that has been appreciated for its neuroprotective properties in ischemic cerebral injuries, yet its intricate mechanisms remain only partially elucidated. AIMS:This study leverages advanced Mass cytometry (CyTOF) to analyze AGNHW's multifaceted immunomodulation effects in-depth, emphasizing previously underexplored areas. RESULTS:AGNHW mitigated monocyte-derived macrophages (MoDM) infiltration in the brain, distinguishing its effects on those from microglia. While the vehicle group exhibited elevated inflammatory markers like CD4, CD8a, and CD44 in ischemic brains, the AGNHW-treated group attenuated their expressions, indicating AGNHW's potential to temper the post-ischemic inflammatory response. Systemically, AGNHW modulated fundamental immune cell dynamics, notably augmenting CD8+ T cells, B cells, monocytes, and neutrophil counts in the peripheral blood under post-stroke conditions. Intracellularly, AGNHW exhibited its targeted modulation of the signaling pathways, revealing a remarked inhibition of key markers like IκBα, indicating potential suppression of inflammatory responses in ischemic brain injuries. CONCLUSION:This study offers a comprehensive portrait of AGNHW's immunomodulation effects on ischemic stroke, illuminating its dual sites of action-both cerebral and systemic-and its nuanced modulation of cellular and molecular dynamics.