Profile of Chinese Cluster Headache Register Individual Study (CHRIS): Clinical characteristics, diagnosis and treatment status data of 816 patients in China.

BACKGROUND: The clinical profile of cluster headache may differ among different regions of the world, warranting interest in the data obtained from the initial Chinese Cluster Headache Register Individual Study (CHRIS) for better understanding. METHODS: We conducted a multicenter, prospective, longitudinal cohort study on cluster headache across all 31 provinces of China, aiming to gather clinical characteristics, treatment approaches, imaging, electrophysiological and biological samples. RESULTS: In total 816 patients were enrolled with a male-to-female ratio of 4.33:1. The mean age at consultation was 34.98 ± 9.91 years, and 24.89 ± 9.77 years at onset. Only 2.33% were diagnosed with chronic cluster headache, and 6.99% had a family history of the condition. The most common bout was one to two times per year (45.96%), lasting two weeks to one month (44.00%), and occurring frequently in spring (76.23%) and winter (73.04%). Of these, 68.50% experienced one to two attacks per day, with the majority lasting one to two hours (45.59%). The most common time for attacks was between 9 am and 12 pm (75.86%), followed by 1 am and 3 am (43.48%). Lacrimation (78.80%) was the most predominant autonomic symptom reported. Furthermore, 39.22% of patients experienced a delay of 10 years or more in receiving a correct diagnosis. Only 35.67% and 24.26% of patients received common acute and preventive treatments, respectively. CONCLUSION: Due to differences in ethnicity, genetics and lifestyle conditions, CHRIS has provided valuable baseline data from China. By establishing a dynamic cohort with comprehensive multidimensional data, it aims to advance the management system for cluster headache in China.

Polyethylenimine Triggers Dll4 Degradation to Regulate Angiogenesis In Vitro.

The Dll4-Notch signaling pathway plays a crucial role in the regulation of angiogenesis and is a promising therapeutic target for diseases associated with abnormal angiogenesis, such as cancer and ophthalmic diseases. Here, we find that polyethylenimine (PEI), a cationic polymer widely used as nucleic acid transfection reagents, can target the Notch ligand Dll4. By immunostaining and immunoblotting, we demonstrate that PEI significantly induces the clearance of cell-surface Dll4 and facilitates its degradation through the lysosomal pathway. As a result, the activation of Notch signaling in endothelial cells is effectively inhibited by PEI, as evidenced by the observed decrease in the generation of the activated form of Notch and expression of Notch target genes Hes1 and Hey1. Furthermore, through blocking Dll4-mediated Notch signaling, PEI treatment enhances angiogenesis in vitro. Together, our study reveals a novel biological effect of PEI and establishes a foundation for the development of a Dll4-targeted biomaterial for the treatment of angiogenesis-related disease.

Integrated transcriptomic and proteomic profiling reveals the key molecular signatures of brain endothelial reperfusion injury.

BACKGROUND: Reperfusion therapy after ischemic stroke often causes brain microvascular injury. However, the underlying mechanisms are unclear. METHODS: Transcriptomic and proteomic analyses were performed on human cerebral microvascular endothelial cells following oxygen-glucose deprivation (OGD) or OGD plus recovery (OGD/R) to identify molecules and signaling pathways dysregulated by reperfusion. Major findings were further validated in a mouse model of cerebral ischemia and reperfusion. RESULTS: Transcriptomic analysis identified 390 differentially expressed genes (DEGs) between the OGD/R and OGD group. Pathway analysis indicated that these genes were mostly associated with inflammation, including the TNF signaling pathway, TGF-ß signaling pathway, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and NF-κB signaling pathway. Proteomic analysis identified 201 differentially expressed proteins (DEPs), which were primarily associated with extracellular matrix destruction and remodeling, impairment of endothelial transport function, and inflammatory responses. Six genes (DUSP1, JUNB, NFKBIA, NR4A1, SERPINE1, and THBS1) were upregulated by OGD/R at both the mRNA and protein levels. In mice with cerebral ischemia and reperfusion, brain TNF signaling pathway was activated by reperfusion, and inhibiting TNF-α with adalimumab significantly attenuated reperfusion-induced brain endothelial inflammation. In addition, the protein level of THBS1 was substantially upregulated upon reperfusion in brain endothelial cells and the peri-endothelial area in mice receiving cerebral ischemia. CONCLUSION: Our study reveals the key molecular signatures of brain endothelial reperfusion injury and provides potential therapeutic targets for the treatment of brain microvascular injury after reperfusion therapy in ischemic stroke.

An MMP-9 exclusive neutralizing antibody attenuates blood-brain barrier breakdown in mice with stroke and reduces stroke patient-derived MMP-9 activity.

Rapid upregulation of matrix metalloproteinase 9 (MMP-9) leads to blood-brain barrier (BBB) breakdown following stroke, but no MMP-9 inhibitors have been approved in clinic largely due to their low specificities and side effects. Here, we explored the therapeutic potential of a human IgG monoclonal antibody (mAb), L13, which was recently developed with exclusive neutralizing specificity to MMP-9, nanomolar potency, and biological function, using mouse stroke models and stroke patient samples. We found that L13 treatment at the onset of reperfusion following cerebral ischemia or after intracranial hemorrhage (ICH) significantly reduced brain tissue injury and improved the neurological outcomes of mice. Compared to control IgG, L13 substantially attenuated BBB breakdown in both types of stroke model by inhibiting MMP-9 activity-mediated degradations of basement membrane and endothelial tight junction proteins. Importantly, these BBB-protective and neuroprotective effects of L13 in wild-type mice were comparable to Mmp9 genetic deletion and fully abolished in Mmp9 knockout mice, highlighting the in vivo target specificity of L13. Meanwhile, ex vivo co-incubation with L13 significantly neutralized the enzymatic activities of human MMP-9 in the sera of ischemic and hemorrhagic stroke patients, or in the peri-hematoma brain tissues from hemorrhagic stroke patients. Overall, we demonstrated that MMP-9 exclusive neutralizing mAbs constitute a potential feasible therapeutic approach for both ischemic and hemorrhagic stroke.

Lithium attenuates blood-brain barrier damage and brain edema following intracerebral hemorrhage via an endothelial Wnt/ß-catenin signaling-dependent mechanism in mice.

BACKGROUND: Vasogenic cerebral edema resulting from blood-brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). Although augmentation of endothelial Wnt/ß-catenin signaling substantially alleviates BBB breakdown in animals, no agents based on this mechanism are clinically available. Lithium is a medication used to treat bipolar mood disorders and can upregulate Wnt/ß-catenin signaling. METHODS: We evaluated the protective effect of lithium on the BBB in a mouse model of collagenase IV-induced ICH. Furthermore, we assessed the effect and dependency of lithium on Wnt/ß-catenin signaling in mice with endothelial deletion of the Wnt7 coactivator Gpr124. RESULTS: Lithium treatment (3 mmol/kg) significantly decreased the hematoma volume (11.15 ± 3.89 mm3 vs. 19.97 ± 3.20 mm3 in vehicle controls, p = 0.0016) and improved the neurological outcomes of mice following ICH. Importantly, lithium significantly increased the BBB integrity, as evidenced by reductions in the levels of brain edema (p = 0.0312), Evans blue leakage (p = 0.0261), and blood IgG extravasation (p = 0.0009) into brain tissue around the hematoma. Mechanistically, lithium upregulated the activity of endothelial Wnt/ß-catenin signaling in mice and increased the levels of tight junction proteins (occludin, claudin-5 and ZO-1). Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, suggesting that its protective effect on BBB function was mainly dependent on Gpr124-mediated endothelial Wnt/ß-catenin signaling. CONCLUSION: Our findings indicate that lithium may serve as a therapeutic candidate for treating BBB breakdown and brain edema following ICH.

Normalization of non-canonical Wnt signalings does not compromise blood-brain barrier protection conferred by upregulating endothelial Wnt/ß-catenin signaling following ischemic stroke.

BACKGROUND: Endothelial canonical (Wnt/ß-catenin) and non-canonical Wnt signalings (Wnt/PCP and Wnt/Ca2+ ) promote blood-brain barrier (BBB) development and antagonize each other. However, the effects of ischemic stroke on endothelial canonical and non-canonical Wnt signalings are unclear. Further, how non-canonical Wnt signalings are influenced by upregulation of endothelial Wnt/ß-catenin signaling and subsequently affect BBB function following ischemic stroke have not been studied. METHODS: First, we determined the levels of Wnt signaling markers including TCF/LEF1 transcription activity, Axin2 mRNA, phospho-JNKThr183/Tyr185 , and NFAT in brain endothelial cells (ECs) with the deletion of Wnt receptor Frizzled (Fzd)4 or Fzd6, the two most abundant Fzds in brain ECs. Next, we observed the effect of ischemia/reperfusion injury on Wnt signalings in brain ECs and adult mice. Last, we assessed the changes of non-canonical Wnt signalings and BBB injury in the early stage of ischemic stroke in mice with endothelial ß-catenin activation (ß-cat mice). RESULTS: Fzd4 or Fzd6 deletion dampened both Wnt/ß-catenin and Wnt/PCP signalings but enhanced Wnt/Ca2+ signaling in brain ECs. Both canonical and non-canonical Wnt signalings in brain ECs were downregulated after ischemia/reperfusion injury in vitro and in vivo. Upregulating endothelial Wnt/ß-catenin signaling in ß-cat mice normalized the downregulated non-canonical Wnt signalings, which did not compromise its protective effects on BBB integrity and endothelial tight junction following ischemic stroke. CONCLUSIONS: The BBB protection induced by upregulation of endothelial Wnt/ß-catenin signaling may be not interfered by the normalization of non-canonical Wnt signalings in the early stage of ischemic stroke.

Lithium alleviates blood-brain barrier breakdown after cerebral ischemia and reperfusion by upregulating endothelial Wnt/ß-catenin signaling in mice.

Although upregulation of endothelial Wnt/ß-catenin signaling may be used to treat blood-brain barrier (BBB) breakdown caused by cerebral ischemia/reperfusion injury, no agents based on this mechanism are available clinically. Lithium, a medication used for treating bipolar mood disorders, upregulates Wnt/ß-catenin signaling, but whether lithium alleviates BBB breakdown after ischemic stroke by upregulating endothelial Wnt/ß-catenin signaling is unclear. Here, we evaluated the BBB-protective effect of lithium in adult mice with 1-h middle cerebral artery occlusion and 48-h reperfusion (MCAO/R) by determining neurological outcomes, BBB function and related molecular components. Furthermore, we assessed the effect and dependence of lithium on Wnt/ß-catenin signaling in brain microvascular endothelial cells in cell culture and in mice with conditional endothelial knockout of Wnt7 co-receptor Gpr124. Our data show that lithium treatment (3 mmol/kg) significantly decreased infarct volume (34.1 ± 1.8% versus 58.3 ± 2.8% in vehicle controls, P < 0.0001) and improved neurological outcomes of mice following MCAO/R. Importantly, lithium significantly increased BBB integrity shown by reduction of Evans blue leakage (by 45.7%, P = 0.0064) and blood IgG extravasation (by 65.8%, P < 0.0001) into infarcted brain tissue. Mechanistically, lithium upregulated the activity of endothelial Wnt/ß-catenin signaling in vivo and in vitro, increased the protein levels of tight junctions (Claudin-5 and ZO-1), and reduced MMP-9 expression. Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, indicating the protection of lithium on BBB was mainly dependent on the Gpr124-mediated endothelial Wnt/ß-catenin signaling. Taken together, our findings indicate that lithium may serve as a therapeutic candidate for treating the BBB breakdown in the early stage of ischemic stroke following reperfusion therapy.

The Combination of Human Urinary Kallidinogenase and Mild Hypothermia Protects Adult Rats Against Hypoxic-Ischemic Encephalopathy-Induced Injury by Promoting Angiogenesis and Regeneration.

Objectives: Human Urinary Kallidinogenase (HUK) is a tissue kallikrein that plays neuroprotective role in ischemic conditions via different mechanisms. Mild hypothermia (MH) is another robust neuroprotectant that reduces mortality but does not profoundly ameliorate the neurological outcome in hypoxic-ischemic encephalopathy (HIE) patients. However, whether the combination of HUK and MH can be used as a promising neuroprotective treatment in HIE is unknown. Methods: One-hundred and forty-four adult Wistar rats were randomly divided into five groups: Sham, HIE, HUK, MH and a combination of HUK and MH treatment. The HIE rat model was established by right carotid dissection followed by hypoxia aspiration. The survival curve was created within 7 days, and the neurological severity scores (NSS) were assessed at days 0, 1, 3, and 7. Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), immunofluorescent staining and western blotting were used to evaluate neuronal survival, apoptosis and necrosis, tight-junction proteins Claudin-1 and Zonula occludens-1 (ZO-1), vascular endothelial growth factor (VEGF), doublecortex (DCX), bradykinin receptor B1 (BDKRB1), BDKRB2 and Ki67 staining. Results: The combined treatment rescued all HIE rats from death and had a best survival curve compared to HIE. The Combination also reduced the NSS scores after HIE at days 7, better than HUK or MH alone. The combination of HUK and MH reserved more cells in Nissl staining and inhibited neuronal apoptosis and necrosis as well as significantly attenuated HIE-induced decreases in claudin-1, ZO-1, cyclin D1 and BDKRB1/B2 in comparison to HUK or MH treatment alone. Moreover, the combined treatment increased the expression of VEGF and DCX as well as the number of Ki67-labeled cells. Conclusions: This study demonstrates that both HUK and MH are neuroprotective after HIE insult; however, the combined therapy with HUK and MH enhanced the efficiency and efficacy of either therapy alone in the treatment of HIE, at least partially by promoting angiogenesis and regeneration and rescuing tight-junction loss. The combination of HUK and MH seems to be a feasible and promising clinical strategy to alleviate cerebral injury following HIE insult. Highlights: -The combination of HUK and MH distinctly reduces neurological dysfunction in HIE rats.-HUK enhances the neuroprotective effects of MH in HIE.-MH attenuates tight-junction disruption, upregulates the BDKR B1/2, DCX and cyclin D1.-The combination of MH and HUK enhances the expressions of MH/HUK mediated-BDKR B1/2, DCX, cyclin D1 and Ki67 positive cells.

TFP5 peptide, derived from CDK5-activating cofactor p35, provides neuroprotection in early-stage of adult ischemic stroke.

Cyclin-dependent kinase 5 (CDK5) is a multifaceted protein shown to play important roles in the central nervous system. Abundant evidence indicates that CDK5 hyperactivities associated with neuronal apoptosis and death following ischemic stroke. CDK5 activity increases when its cofactor p35 cleaves into p25 during ischemia. Theoretically, inhibition of CDK5/p25 activity or reduction of p25 would be neuroprotective. TFP5, a modified 24-aa peptide (Lys254-Ala277) derived from p35, was found to effectively inhibit CDK5 hyperactivity and improve the outcomes of Alzheimer's disease and Parkinson's disease in vivo. Here, we showed that intraperitoneal injection of TFP5 significantly decreased the size of ischemia in early-stage of adult ischemic stroke rats. Relative to controls, rats treated with TFP5 displayed reduced excitotoxicity, neuroinflammation, apoptosis, astrocytes damage, and blood-brain barrier disruption. Our findings suggested that TFP5 might serve as a potential therapeutic candidate for acute adult ischemic stroke.

TFP5 is comparable to mild hypothermia in improving neurological outcomes in early-stage ischemic stroke of adult rats.

AIM: We compared the efficacy of a modified truncated 24-aa peptide (TFP5), derived from the cyclin-dependent kinase 5 (CDK5)-activating cofactor p35, with mild hypothermia (MH), and determined whether the efficacy of TFP5 is affected by MH. METHODS: Ischemic stroke was induced in adult male Sprague-Dawley rats for 2h. Immediately after initiating reperfusion, TFP5, MH, or the combination of the two were administrated. 48h after reperfusion, neurological outcomes were evaluated. RESULTS: Rats that received either MH, TFP5, or the combined treatment showed smaller brain infarct size than normothermia control (NT), and there was no apparent difference among these three treatment groups. The neurological deficit was significantly improved only by the combined treatment. MH or TFP5 ameliorated the blood-brain barrier (BBB) disruption in ischemic regions with similar efficacy, whereas the combination of them had a trend toward better effect. Besides, the cleavage of p35 into p25 and apoptosis in ischemic regions was inhibited by TFP5 or the combination, but not by MH alone. CONCLUSIONS: TFP5 is comparable to MH in improving neurological outcomes in early-stage adult ischemic stroke. When TFP5 is given along with MH, less neurological deficit tends to be achieved.

Intra-carotid cold magnesium sulfate infusion induces selective cerebral hypothermia and neuroprotection in rats with transient middle cerebral artery occlusion.

Local hypothermia induced by intra-arterial infusion of cold saline reduces brain injury in ischemic stroke. Administration of magnesium sulfate through the internal carotid artery is also known to reduce ischemic brain damage. The neuroprotective effects of combination therapy with local endovascular hypothermia and intra-carotid magnesium sulfate infusion has not been evaluated. The aim of the study was to determine whether infusion of intra-carotid cold magnesium offers neuroprotective efficacy superior to cold saline infusion alone. Sixty-eight Sprague-Dawley rats were subjected to 3 h of middle cerebral artery occlusion and were randomly divided into six groups: sham-operated group; stroke control group; local cold magnesium infusion group; local cold saline infusion group; local normothermic magnesium infusion group; and local normothermic saline infusion group. Before reperfusion, ischemic rats received local infusion or no treatment. Infarct volume, neurological deficit, and brain water content were evaluated at 48 h after reperfusion. Selective brain hypothermia (33-34 °C) was successfully induced by intra-carotid cold infusion. Local cold saline infusion and local cold magnesium infusion reduced the infarct volumes by 48 % (p < 0.001) and 65 % (p < 0.001), respectively, compared with stroke controls. Brain water content was decreased significantly in animals treated with local cold magnesium infusion. Furthermore, the rats given a local cold magnesium infusion had the best neurological outcome. Local normothermic infusion failed to improve ischemic brain damage. These data suggest that local hypothermia induced by intra-carotid administration of cold magnesium is more effective in reducing acute ischemic damage than infusion of cold saline alone.

Interrupted intracarotid artery cold saline infusion as an alternative method for neuroprotection after ischemic stroke.

OBJECT: Intracarotid artery cold saline infusion (ICSI) is an effective method for protecting brain tissue, but its use is limited because of undesirable secondary effects, such as severe decreases in hematocrit levels, as well as its relatively brief duration. In this study, the authors describe and investigate the effects of a novel ICSI pattern (interrupted ICSI) relative to the traditional method (uninterrupted ICSI). METHODS: Ischemic strokes were induced in 85 male Sprague-Dawley rats by occluding the middle cerebral artery for 3 hours using an intraluminal filament. Uninterrupted infusion groups received an infusion at 15 ml/hour for 30 minutes continuously. The same infusion speed was used in the interrupted infusion groups, but the whole duration was divided into trisections, and there was a 20-minute interval without infusion between sections. Forty-eight hours after reperfusion, H & E and silver nitrate staining were utilized for morphological assessment. Infarct sizes and brain water contents were determined using H & E staining and the dry-wet weight method, respectively. Levels of neuron-specific enolase (NSE), S100ß protein, and matrix metalloproteinase 9 (MMP-9) in the serum were determined using enzyme-linked immunosorbent assay. Neurological deficits were also evaluated. RESULTS: Histology showed that interrupted ICSI did not affect neurons or fibers in rat brains, which suggests that this method is safe for brain tissues with ischemia. The duration of hypothermia induced by interrupted ICSI was longer than that induced via the traditional method, and the decrease in hematocrit levels was less pronounced. There were no differences in infarct size or brain water content between uninterrupted and interrupted ICSI groups, but neuron-specific enolase and matrix metalloproteinase 9 serum levels were more reduced after interrupted ICSI than after the traditional method. CONCLUSIONS: Interrupted ICSI is a safe method. Compared with traditional ICSI, the interrupted method has a longer duration of hypothermia and less effect on hematocrit and offers more potentially improved neuroprotection, thereby making it more attractive as an infusion technique in the clinic.

Therapeutic time window of hypothermia is broader than cerebral artery flushing in carotid saline infusion after transient focal ischemic stroke in rats.

OBJECT: Intracarotid cold saline infusion (ICSI) protects against ischemic stroke not only due to the resulting hypothermia, but also as a result of the cerebral artery flushing. To assess the relative benefit of hypothermia and cerebral artery flushing in neuroprotection, hypothermic and normothermic saline infusions were administrated over a serial time points after the initiation of reperfusion in a rat ischemia model. METHODS: Ischemic strokes were induced in Sprague-Dawley rats (n = 115) by occluding the middle cerebral artery for 2 hours using an intraluminal filament. In the hypothermic groups, the brain temperature was lowered to 33-34°C for 20 minutes by ICSI at three time points (0, 1, and 2 hours) after reperfusion. Correspondingly, in the normothermic groups, the brain temperature was maintained at normal levels during intracarotid normothermic saline infusion (INSI) for 20 minutes at the same time points. After 48-hour reperfusion, infarct sizes and brain water contents were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining and the dry-wet weight method, respectively. Levels of neuron-specific enolase (NSE), S100beta, and matrix metalloproteinase 9 (MMP9) in the serum were measured by enzyme-linked immunoassay (ELISA). Neurological deficits were also evaluated. RESULTS: Immediate infusion after the onset of reperfusion (0 hour) did not result in significant difference for reductions of infarct sizes, neurological deficits or S100beta serum levels between ICSI and INSI groups, compared with the non-infusion group. However, brain water content and NSE serum level were significantly lower in the ICSI group than the non-infusion group. When the infusions were started 1 hour after reperfusion, both ICSI and INSI infusions still reduced the infarct sizes, but only ICSI significantly decreased the brain water content, neurological deficits and S100beta serum level. All therapeutic effects of INSI disappeared when infusions were started 2 hours after reperfusion, whereas infarct size, neurological deficits and S100beta serum level were still reduced significantly in ICSI group, compared with the non-infusion group. CONCLUSIONS: The neuroprotection of hypothermia and cerebral artery flushing induced by selective carotid infusion after ischemia weakens as the length of time between the reperfusion and infusion increases. The therapeutic time window of brain hypothermia induced by cold saline infusion is broader than cerebral artery flushing induced by normothermic saline infusion.

[Comparison of neuroprotective effects of hypothermia induced by different methods in rats with early cerebral ischemia].

OBJECTIVE: To compare the neuroprotective effects of intracarotid cold saline infusion (ICSI), ice cap and systemic cooling in rats with early cerebral ischemia. METHODS: SD rats were randomly divided into model group, ice cap group, systemic cooling group and ICSI group (n=13). Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h. In the 3 hypothermic groups, the target temperature of 33-34 degrees celsius; was achieved and maintained for 20 min. In each group, the neurological scores were recorded at 48 h after the reperfusion, and the brains were removed for measuring the infarct size using 2,3,5-triphenyltetrazolium chloride staining. The brain water contents and serum levels of neuron-specific enolase (NSE), S100ß and matrix metalloproteinase 9 (MMP9) were also measured. RESULTS: Compared with the model group, the ice cap, systemic cooling and ICSI groups all showed significantly reduced infarct size by 27.4% (P<0.05), 47.6% (P<0.01) and 61.6% (P<0.01), respectively. The systemic cooling and ICSI groups, but not the ice cap group, had significantly lower brain water contents than the model group (P<0.05). Among the 3 hypothermic methods, only ICSI significantly improved the neurological scores (P<0.05) and reduced serum NSE and S100ß levels (P<0.05). CONCLUSION: Of the 3 hypothermic groups, ICSI has the best neuroprotective effects, and systemic cooling produces better effect than ice cap in rats with ischemic stroke.