GPR39 Agonist TC-G 1008 Promoted Mitochondrial Biogenesis and Improved Antioxidative Capability via CREB/PGC-1α Pathway Following Intracerebral Hemorrhage in Mice.

Mitochondrial dysfunction and excessive reactive oxygen species production due to impaired mitochondrial biogenesis have been proven to exacerbate secondary brain injury after intracerebral hemorrhage (ICH). The G-protein-coupled receptor 39 (GPR39) agonist TC-G 1008 has been shown to exert anti-oxidative stress effect in acute hypoxic brain injury. Herein, our study aimed to investigate the potential effects of TC-G 1008 on neuronal mitochondrial biogenesis and antioxidative stress in a mouse model of ICH and explore the underlying mechanisms. A total of 335 male C57/BL6 mice were used to establish an autologous blood-induced ICH model. Three different dosages of TC-G 1008 were administered via oral gavage at 1 h, 25 h, and 49 h post-ICH. The GPR39 siRNA and cAMP response element-binding protein (CREB) inhibitor 666-15 were administered via intracerebroventricular injection before ICH insult to explore the underlying mechanisms. Neurobehavioral function tests, Western blot, quantitative polymerase chain reaction, immunofluorescence staining, Fluoro-Jade C staining, TUNEL staining, dihydroethidium staining, transmission electron microscopy, and enzyme-linked immunosorbent assay were performed. Expression of endogenous GPR39 gradually increased in a time-dependent manner in the peri-hematoma tissues, peaking between 24 and 72 h after ICH. Treatment with TC-G 1008 significantly attenuated brain edema, hematoma size, neuronal degeneration, and neuronal death, as well as improved neurobehavioral deficits at 72 h after ICH. Moreover, TC-G 1008 upregulated the expression of mitochondrial biogenesis-related molecules, including PGC-1α, NRF1, TFAM, and mitochondrial DNA copy number, associated with antioxidative stress markers, such as Nrf2, HO-1, NQO1, SOD, CAT, and GSH-Px. Furthermore, treatment with TC-G 1008 preserved neuronal mitochondrial function and structure post-ICH. Mechanistically, the protective effects of TC-G 1008 on neuronal mitochondrial biogenesis and antioxidative stress were partially reversed by GPR39 siRNA or 666 -15. Our findings indicated that GPR39 agonist TC-G 1008 promoted mitochondrial biogenesis and improved antioxidative capability after ICH, partly through the CREB/PGC-1α signaling pathway. TC-G 1008 may be a potential therapeutic agent for patients with ICH.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

BACKGROUD: Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. METHODS: To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. RESULTS: We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. CONCLUSIONS: The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

Mitochondrial ferritin upregulation reduced oxidative stress and blood-brain-barrier disruption by maintaining cellular iron homeostasis in a neonatal rat model of germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a devasting neurological disease in premature newborns. After GMH, brain iron overload associated with hemoglobin degradation contributed to oxidative stress, causing disruption of the already vulnerable blood-brain barrier (BBB). Mitochondrial ferritin (FTMT), a novel mitochondrial outer membrane protein, is crucial in maintaining cellular iron homeostasis. We aimed to investigate the effect of FTMT upregulation on oxidative stress and BBB disruption associated with brain iron overload in rats. A total of 222 Sprague-Dawley neonatal rat pups (7 days old) were used to establish a collagenase-induced GMH model and an iron-overload model of intracerebral FeCl2 injection. Deferiprone was administered via gastric lavage 1 h after GMH and given daily until euthanasia. FTMT CRISPR Knockout and adenovirus (Ad)-FTMT were administered intracerebroventricularly 48 h before GMH and FeCl2 injection, respectively. Neurobehavioral tests, immunofluorescence, Western blot, Malondialdehyde measurement, and brain water content were performed to evaluate neurobehavior deficits, oxidative stress, and BBB disruption, respectively. The results demonstrated that brain expressions of iron exporter Ferroportin (FPN) and antioxidant glutathione peroxidase 4 (GPX4) as well as BBB tight junction proteins including Claudin-5 and Zona Occulta (ZO)-1 were found to be decreased at 72 h after GMH. FTMT agonist Deferiprone attenuated oxidative stress and preserved BBB tight junction proteins after GMH. These effects were partially reversed by FTMT CRISPR Knockout. Iron overload by FeCl2 injection resulted in oxidative stress and BBB disruption, which were improved by Ad-FTMT mediated FTMT overexpression. Collectively, FTMT upregulation is neuroprotective against brain injury associated with iron overload. Deferiprone reduced oxidative stress and BBB disruption by maintaining cellular iron homeostasis partially by the upregulating of FTMT after GMH. Deferiprone may be an effective treatment for patients with GMH.

Procalcitonin/Albumin Ratio Predicts the Outcome After Severe Traumatic Brain Injury: A Propensity Score-Matched Analysis.

BACKGROUND: The procalcitonin/albumin ratio (PAR), a novel inflammation-based index, has been reported to predict the prognosis following cardiopulmonary bypass surgery and bacterial infection. However, whether PAR can predict the outcome of patients with severe traumatic brain injury (STBI) has not been fully elucidated. This study aimed to investigate the relationship between serum PAR levels and prognosis at 6 months after STBI. METHODS: We retrospectively enrolled 129 patients diagnosed with STBI and collected relevant clinical and laboratory data. Logistic regression analysis was used to estimate the association of PAR with the prognosis of STBI. The receiver operating characteristics curve was performed to examine the predictive use of PAR for prognosis. Propensity score matching (PSM) analysis was also performed to improve the reliability of the results. The primary outcome measures were expressed as a score on the modified Rankin Scale at 6 months. RESULTS: The unfavorable prognosis group had advanced age, lower Glasgow Coma Scale score, higher rate of cerebral hernia and intracranial infection, higher neutrophil/lymphocyte ratio (NLR) and C-reactive protein/albumin ratio (CAR), elevated PAR, and higher rate of pneumonia. Multivariate analysis showed that PAR (before PSM: odds ratio 3.473, 95% confidence interval 2.983-4.043, P < 0.001; after PSM: odds ratio 5.358, 95% confidence interval 3.689-6.491, P < 0.001) was independently associated with unfavorable outcome. The area under the curve of the PAR for predicting an unfavorable outcome was higher than that of the CAR and NLR. CONCLUSIONS: The PAR might be a novel independent risk factor of the outcome after STBI. Moreover, PAR was a better biomarker in predicting the outcome of patients with STBI than CAR and NLR.

Integrating network pharmacology and transcriptomic omics reveals that akebia saponin D attenuates neutrophil extracellular traps-induced neuroinflammation via NTSR1/PKAc/PAD4 pathway after intracerebral hemorrhage.

Neutrophils and their production of neutrophil extracellular traps (NETs) significantly contribute to neuroinflammation and brain damage after intracerebral hemorrhage (ICH). Although Akebia saponin D (ASD) demonstrates strong anti-inflammatory activities and blood-brain barrier permeability, its role in regulating NETs formation and neuroinflammation following ICH is uncharted. Our research focused on unraveling the influence of ASD on neuroinflammation mediated by NETs and the mechanisms involved. We found that increased levels of peripheral blood neutrophils post-ICH are correlated with worse prognostic outcomes. Through network pharmacology, we identified ASD as a promising therapeutic target for ICH. ASD administration significantly improved neurobehavioral performance and decreased NETs production in neutrophils. Furthermore, ASD was shown to upregulate the membrane protein NTSR1 and activate the cAMP signaling pathway, confirmed through transcriptome sequencing, western blot, and immunofluorescence. Interestingly, the NTSR1 inhibitor SR48692 significantly nullified ASD's anti-NETs effects and dampened cAMP pathway activation. Mechanistically, suppression of PKAc via H89 negated ASD's anti-NETs effects but did not affect NTSR1. Our study suggests that ASD may reduce NETs formation and neuroinflammation, potentially involving the NTSR1/PKAc/PAD4 pathway post-ICH, underlining the potential of ASD in mitigating neuroinflammation through its anti-NETs properties.

Insight into the Progress in CAR-T Cell Therapy and Combination with Other Therapies for Glioblastoma.

Glioblastoma (GBM) is the most common malignant primary brain cancer in adults. It is always resistant to existing treatments, including surgical resection, postoperative radiotherapy, and chemotherapy, which leads to a dismal prognosis and a high relapse rate. Therefore, novel curative therapies are urgently needed for GBM. Chimeric antigen receptor T (CAR-T) cell therapy has significantly improved life expectancy for hematological malignancies patients, and thus it increases the interest in applying CAR-T cell therapy for solid tumors. In the recently published research, it is indicated that there are numerous obstacles to achieve clinical benefits for solid tumors, especially for GBM, because of GBM anatomical characteristics (the blood-brain barrier and suppressive tumor microenvironment) and the tumor heterogeneity. CAR-T cells are difficult to penetrate blood-brain barrier, and immunosuppressive tumor microenvironment (TME), which induces CAR-T cell exhaustion, impairs CAR-T cell therapy response. Moreover, under the pressure of CAR-T cell therapy, the tumor heterogeneity and tumor plasticity drive tumor evolution and therapy resistance, such as antigen escape. Nonetheless, scientists strive for strategies to overcome these hurdles, including novel CAR-T cell designs and regional delivery. For instance, the structure of multi-antigen-targeted CAR-T cells can enrich CAR-T accumulation in tumor TME and eliminate abundant tumor cells to avoid tumor antigen heterogeneity. Additionally, paired with an immune modifier and one or more stimulating domains, different generation of innovations in the structure and manufacturing of CAR-T cells have improved efficacy and persistence. While single CAR-T cell therapy receives limited clinical survival benefit. Compared with single CAR-T cell therapy, the combination therapies have supplemented the treatment paradigm. Combinatorial treatment methods consolidate the CAR-T cells efficacy by regulating the tumor microenvironment, optimizing the CAR structure, targeting the CAR-T cells to the tumor cells, reversing the tumor-immune escape mechanisms, and represent a promising avenue against GBM, based on multiple impressive research. Moreover, exciting results are also reported to be realized through combining effective therapies with CAR-T cells in preclinical and clinical trials samples, have aroused inspiration to explore the antitumor function of combination therapies. In summary, this study aims to summarize the limitation of CAR-T cell therapies and introduces novel strategies to enhance CAR-T cell function as well as prospect the potential of the therapeutic combination.

Histological and molecular glioblastoma, IDH-wildtype: a real-world landscape using the 2021 WHO classification of central nervous system tumors.

Introduction: Glioblastoma (GBM), the most lethal primary brain malignancy, is divided into histological (hist-GBM) and molecular (mol-GBM) subtypes according to the 2021 World Health Organization classification of central nervous system tumors. This study aimed to characterize the clinical, radiological, molecular, and survival features of GBM under the current classification scheme and explore survival determinants. Methods: We re-examined the genetic alterations of IDH-wildtype diffuse gliomas at our institute from 2011 to 2022, and enrolled GBMs for analysis after re-classification. Univariable and multivariable analyses were used to identify survival determinants. Results: Among 209 IDH-wildtype gliomas, 191 were GBMs, including 146 hist-GBMs (76%) and 45 mol-GBMs (24%). Patients with mol-GBMs were younger, less likely to develop preoperative motor dysfunction, and more likely to develop epilepsy than hist-GBMs. Mol-GBMs exhibited lower radiographic incidences of contrast enhancement and intratumoral necrosis. Common molecular features included copy-number changes in chromosomes 1, 7, 9, 10, and 19, as well as alterations in EGFR, TERT, CDKN2A/B, and PTEN, with distinct patterns observed between the two subtypes. The median overall survival (mOS) of GMB was 12.6 months. Mol-GBMs had a higher mOS than hist-GBMs, although not statistically significant (15.6 vs. 11.4 months, p=0.17). Older age, male sex, tumor involvement of deep brain structure or functional area, and genetic alterations in CDK4, CDK6, CIC, FGFR3, KMT5B, and MYB were predictors for a worse prognosis, while MGMT promoter methylation, maximal tumor resection, and treatment based on the Stupp protocol were predictive for better survival. Conclusion: The definition of GBM and its clinical, radiological, molecular, and prognostic characteristics have been altered under the current classification.

An online survival predictor in glioma patients using machine learning based on WHO CNS5 data.

Background: The World Health Organization (WHO) CNS5 classification system highlights the significance of molecular biomarkers in providing meaningful prognostic and therapeutic information for gliomas. However, predicting individual patient survival remains challenging due to the lack of integrated quantitative assessment tools. In this study, we aimed to design a WHO CNS5-related risk signature to predict the overall survival (OS) rate of glioma patients using machine learning algorithms. Methods: We extracted data from patients who underwent an operation for histopathologically confirmed glioma from our hospital database (2011-2022) and split them into a training and hold-out test set in a 7/3 ratio. We used biological markers related to WHO CNS5, clinical data (age, sex, and WHO grade), and prognosis follow-up information to identify prognostic factors and construct a predictive dynamic nomograph to predict the survival rate of glioma patients using 4 kinds machine learning algorithms (RF, SVM, XGB, and GLM). Results: A total of 198 patients with complete WHO5 molecular data and follow-up information were included in the study. The median OS time of all patients was 29.77 [95% confidence interval (CI): 21.19-38.34] months. Age, FGFR2, IDH1, CDK4, CDK6, KIT, and CDKN2A were considered vital indicators related to the prognosis and OS time of glioma. To better predict the prognosis of glioma patients, we constructed a WHO5-related risk signature and nomogram. The AUC values of the ROC curves of the nomogram for predicting the 1, 3, and 5-year OS were 0.849, 0.835, and 0.821 in training set, and, 0.844, 0.943, and 0.959 in validation set. The calibration plot confirmed the reliability of the nomogram, and the c-index was 0.742 in training set and 0.775 in validation set. Additionally, our nomogram showed a superior net benefit across a broader scale of threshold probabilities in decision curve analysis. Therefore, we selected it as the backend for the online survival prediction tool (Glioma Survival Calculator, https://who5pumch.shinyapps.io/DynNomapp/), which can calculate the survival probability for a specific time of the patients. Conclusion: An online prognosis predictor based on WHO5-related biomarkers was constructed. This therapeutically promising tool may increase the precision of forecast therapy outcomes and assess prognosis.

CDGSH iron sulfur domain 2 over-expression alleviates neuronal ferroptosis and brain injury by inhibiting lipid peroxidation via AKT/mTOR pathway following intracerebral hemorrhage in mice.

Ferroptosis has been implicated in the pathogenesis of secondary brain injury following intracerebral hemorrhage (ICH), and regulating this process is considered a potential therapy for alleviating further brain injury. A previous study showed that CDGSH iron sulfur domain 2 (CISD2) can inhibit ferroptosis in cancer. Thus, we investigated the effects of CISD2 on ferroptosis and the mechanisms underlying its neuroprotective role in mice after ICH. CISD2 expression markedly increased after ICH. CISD2 over-expression significantly decreased the number of Fluoro-Jade C-positive neurons and alleviated brain edema and neurobehavioral deficits at 24 h after ICH. In addition, CISD2 over-expression up-regulated the expression of p-AKT, p-mTOR, ferritin heavy chain 1, glutathione peroxidase 4, ferroportin, glutathione, and glutathione peroxidase activity, which are markers of ferroptosis. Additionally, CISD2 over-expression down-regulated the levels of malonaldehyde, iron content, acyl-CoA synthetase long-chain family member 4, transferrin receptor 1, and cyclooxygenase-2 at 24 h after ICH. It also alleviated mitochondrial shrinkage and decreased the density of the mitochondrial membrane. Furthermore, CISD2 over-expression increased the number of GPX4-positive neurons following ICH induction. Conversely, knockdown of CISD2 aggravated neurobehavioral deficits, brain edema, and neuronal ferroptosis. Mechanistically, MK2206, an AKT inhibitor, suppressed p-AKT and p-mTOR and reversed the effects of CISD2 over-expression on markers of neuronal ferroptosis and acute neurological outcome. Taken together, CISD2 over-expression alleviated neuronal ferroptosis and improved neurological performance, which may be mediated through the AKT/mTOR pathway after ICH. Thus, CISD2 may be a potential target to mitigate brain injury via the anti-ferroptosis effect after ICH.

Microglial pyroptosis: Therapeutic target in secondary brain injury following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a major cerebrovascular illness that causes substantial neurological sequelae and dysfunction caused by secondary brain injury (SBI), and there are no effective therapies to mitigate the disability. Microglia, the brain-resident macrophage, participates in the primary inflammatory response, and activation of microglia to an M1-like phenotype largely takes place in the acute phase following ICH. A growing body of research suggests that the pathophysiology of SBI after ICH is mediated by an inflammatory response mediated by microglial-pyroptotic inflammasomes, while inhibiting the activation of microglial pyroptosis could suppress the inflammatory cascade reaction, thus attenuating the brain injury after ICH. Pyroptosis is characterized by rapid plasma membrane disruption, followed by the release of cellular contents and pro-inflammatory mediators. In this review, we outline the molecular mechanism of microglial pyroptosis and summarize the up-to-date evidence of its involvement in the pathological process of ICH, and highlight microglial pyroptosis-targeted strategies that have the potential to cure intracerebral hemorrhage. This review contributes to a better understanding of the function of microglial pyroptosis in ICH and assesses it as a possible therapeutic target.

Activation of RKIP Binding ASC Attenuates Neuronal Pyroptosis and Brain Injury via Caspase-1/GSDMD Signaling Pathway After Intracerebral Hemorrhage in Mice.

Pyroptosis has been proven to be responsible for secondary brain injury after intracerebral hemorrhage (ICH). A recent study reported that Raf kinase inhibitor protein (RKIP) inhibited assembly and activation of inflammasome in macrophages. Our present study aimed to investigate the effects of RKIP on inflammasome-mediated neuronal pyroptosis and underlying neuroprotective mechanisms in experimental ICH. Here, we showed that RKIP expression was decreased both in cerebrospinal fluid (CSF) samples from patients with ICH and in the peri-hematoma tissues after experimental ICH. In mouse ICH model, activation of RKIP remarkably improved neurological deficits, reduced brain water content and BBB disruption, and promoted hematoma absorption at 24 h after ICH, as well as alleviated neuronal degeneration, reduced membrane pore formation, and downregulated pyroptotic molecules NLRP3, caspase-1 P20, GSDMD-N, and mature IL-1ß. Besides, RKIP activation decreased the number of caspase-1 P20-positive neurons after ICH. However, RKIP inhibitor reserved the neuroprotective effects of RKIP at 24 h following ICH. Moreover, RKIP could bind with ASC, then interrupt the assembly of NLRP3 inflammasome. Mechanistically, inhibiting the caspase-1 by VX-765 attenuated brain injury and suppressed neuronal pyroptosis after RKIP inhibitor-pretreated ICH. In conclusion, our findings indicated that activation of RKIP could attenuate neuronal pyroptosis and brain injury after ICH, to some extent, through ASC/Caspase-1/GSDMD pathway. Thus, RKIP may be a potential target to attenuate brain injury via its anti-pyroptosis effect after ICH.

Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage.

Neuroinflammation has been proven to exert an important effect on brain injury after intracerebral hemorrhage (ICH). Previous studies reported that Didymin possessed anti-inflammatory properties after acute hepatic injury, hyperglycemia-induced endothelial dysfunction, and death. However, the role of Didymin in microglial pyroptosis and neuroinflammation after ICH is unclear. The current study aimed to investigate the effect of Didymin on neuroinflammation mediated by microglial pyroptosis in mouse models of ICH and shed some light on the underlying mechanisms. In this study, we observed that Didymin treatment remarkably improved neurobehavioral performance and decreased BBB disruption and brain water content. Microglial activation and neutrophil infiltration in the peri-hematoma tissue after ICH were strikingly mitigated by Didymin as well. At the molecular level, administration of Didymin significantly unregulated the expression of Rkip and downregulated the expression of pyroptotic molecules and inflammatory cytokines such as Nlrp3 inflammasome, GSDMD, caspase-1, and mature IL-1ß, TNF-α, and MPO after ICH. Besides, Didymin treatment decreased the number of Caspase-1-positive microglia and GSDMD-positive microglia after ICH. Inversely, Locostatin, an Rkip-specific inhibitor, significantly abolished the anti-pyroptosis and anti-neuroinflammation effects of Didymin. Moreover, Rkip binding with Asc could interrupt the activation and assembly of the inflammasome. Mechanistically, inhibition of Caspase-1 by VX-765 attenuated brain injury and suppressed microglial pyroptosis and neuroinflammation by downregulation of GSDMD, mature IL-1ß, TNF-α, and MPO based on Locostatin-treated ICH. Taken together, Didymin alleviated microglial pyroptosis and neuroinflammation, at least in part through the Asc/Caspase-1/GSDMD pathway via upregulating Rkip expression after ICH. Therefore, Didymin may be a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.

Improvement of cell counting method for Neubauer counting chamber.

BACKGROUND: We compared the cell counting accuracy of the conventional method and the improved method by using Neubauer counting chamber. METHODS: In the improved method, all the border cells were counted and then divided by two; while, in the conventional method, only border cells on the two boundaries (top and left) were counted. RESULTS: About 55.814% of the samples showed more accurate results by improved counting method, about 38.372% had more accurate results by conventional counting method, and about 5.814% were counted with similar counting error by both methods. The improved method had significantly smaller counting error than conventional method (P < .05). The distribution ratio of the border cells was an independent factor for counting accuracy (P < .05). CONCLUSION: Together, the improved counting method can reduce the counting error of the Neubauer counting chamber to some extent, assess the distributing uniformity of border cells, and help to eliminate the samples with large differences in distribution.

Cardiorespiratory fitness as a quantitative predictor of the risk of stroke: a dose-response meta-analysis.

BACKGROUND AND PURPOSE: Cardiorespiratory fitness (CRF) is closely related to our health, but whether high cardiorespiratory fitness could reduce stroke risk remains controversial. We used a meta-analysis to examine the overall association between cardiorespiratory fitness and stroke incidence. METHODS: A systematic search was performed in PubMed, EMBASE, and Web of science. We estimated the overall relative risk (RR) of stroke incidence for high levels of CRF individuals versus individuals with low levels of CRF. Meanwhile, we made a quantitative analysis of the association between CRF and the risk of stroke. RESULTS: 14 cohort studies containing 1,409,340 participants were included in this meta-analysis, and 23,894 stroke patients were observed. The meta-analysis documented that the high-CRF individuals had a 42% lower risk of stroke (RR_0.58; 95% CI 0.51-0.66) compared with low-CRF individuals. Besides, subgroup analysis showed that the inverse association of CRF with the risk of stroke was consistent. There are 29% lower risk of ischemic stroke (RR_0.71; 95% CI 0.54-0.93) and 31% lower risk of hemorrhagic stroke (RR_0.69; 95% CI 0.47-1.00). Dose-response analysis showed that every "unit-dose" increment of five metabolic equivalents (METs) reduced a 15% lower risk of stroke (RR_0.85; 95% CI 0.79-0.91). CONCLUSION: This meta-analysis provides the evidence that better CRF was with a lower risk of stroke incidence.