Clinical Significance of DKK2 Protein in Cerebral Ischemia-reperfusion Injury

Study Overview
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Brief Summary
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The study is a two-center prospective cohort clinical trial. The primary purpose of this trial is to identify the pattern of DKK2 serum levels in ischemic stroke patients after revascularization therapy and determine the correlation between serum DKK2 levels and prognosis.

Detailed Description
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Ischemic stroke refers to ischaemic and hypoxic necrosis of brain tissue caused by narrowing or occlusion of the blood vessels in the brain and accounts for approximately 80% of all strokes. It is characterized by high morbidity, mortality, disability, and recurrence rates. Reperfusion is currently the most effective treatment for the acute phase of ischaemic stroke, including pharmacological thrombolysis and mechanical embolization. Although successful revascularization can reperfuse areas of cerebral ischemia, it can cause acute cerebrovascular damage while restoring blood supply to brain tissue, leading to disruption of the blood-brain barrier (BBB), increased risk of cerebral edema and hemorrhagic transformation, and increased inflammation of neural tissue, which can further damage brain tissue. Targeted reduction of endothelial damage from ischemia-reperfusion will therefore effectively protect neurons from subsequent damage, thereby minimizing neurological impairment after stroke and maximizing the benefit of revascularisation therapy. The Wnt signaling pathway has been identified by several research groups worldwide as a key regulatory pathway in the maintenance of cerebrovascular and neural cell function. The DKK (Dickkopf-related protein) family of proteins is the most representative group of classical Wnt signaling pathway inhibitors. DKK proteins competitively bind to the Wnt co-receptor LRP5/6, thereby inhibiting the activity of Wnt proteins and exerting their inhibitory effects on the Wnt/β-catenin signaling pathway. We initially found that DKK2 serum levels increased significantly after 4.5 h of recanalization therapy in 20 patients with large vessel occlusive acute stroke, and decreased after 24 h. Increased DKK2 levels were strongly associated with an unfavorable prognosis. This was corroborated in animal models as well, DKK2 expression levels in ischaemic brain tissue and peripheral blood were both significantly elevated and rapidly upregulated within 6-12 h of the onset of cerebral ischemia-reperfusion in mice. In vitro cellular assays showed that DKK2 protein significantly inhibited the activity of the Wnt/β-catenin signaling pathway. We further study showed that upregulation of DKK2 protein levels in the blood of mice by intravenous administration of adenovirus expressing DKK2 protein significantly increased cerebral infarction and neurological impairment in mice with stroke. The increased expression of DKK2 protein in brain tissue is the main reason for the downregulation of Wnt/β-catenin signaling pathway activity after ischemia/reperfusion, which leads to blood-brain barrier damage, neuronal cell death, and neuroinflammation, and ultimately promotes brain tissue damage and neurological dysfunction. It is a new target for drug therapy and has great scientific significance and clinical application prospects. This clinical study is conducted at Dongguan Hospital of Southern Medical University and Nanfang Hospital of Southern Medical University. Acute ischemic stroke patients with large vessel occlusion who received mechanical thrombectomy therapy and are successfully revascularized will be included and followed up for 90 d, along with testing serum levels of DKK2 protein to explore its correlation with the prognosis of enrolled patients. Venous blood samples will be collected before, and 24 h, 48 h, and 72 h after revascularization treatment in enrolled patients. Venous blood samples will be collected before and 0h, 24 h, 48 h, and 72 h after revascularisation treatment to test serum KKD2 level, and cranial CT examination will be performed before, 24 h, and 72 h after revascularization treatment to detect the occurrence of the transformation of hemorrhage, the severity of cerebral edema, and midline shift after revascularisation treatment. Blood-brain barrier injury-related indicators (MMP-9, ICAM-1) and inflammation-related indicators (IL-6, IL-1β, TNF-α, IL-10) will be measured at each time point of DKK2 testing. NIHSS scores will be evaluated before, 0h (immediately after revascularization treatment), 24 h, 48 h, 72 h, and 7 d after revascularization treatment. The mRS scores will be followed up at 30 days and 90 days after the onset to clarify the relationship between serum DKK2 levels and large vessel occlusion. We aim to investigate the mechanism of DKK2 causing adverse clinical outcomes such as BBB leakage, cerebral edema, and hemorrhagic transformation at a real-world clinical level by collecting blood samples, clinical follow-up, and neurological scoring from stroke patients by measuring DKK2 levels and brain imaging parameters for quantitative assessment.

Official Title
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Clinical Significance, Mechanism of Action, and New Targeted Drug Research of DKK2 Protein in Cerebral Ischemia-reperfusion Injury

Conditions
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Ischemic Stroke, Acute, Ischemia-reperfusion Injury, Cerebral Edema

Intervention / Treatment
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* Diagnostic Test: enzyme-linked immunosorbent assay(ELISA)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age ≥ 18 years, < 80 years, sex not limited; Definite clinical diagnosis of acute ischemic stroke; Baseline NIHSS score ≥ 6 and ≤ 25; CTA/MRA/DSA examination suggests large vessel occlusion in the anterior circulation (internal carotid artery, M1/M2 segment of the middle cerebral artery); The criteria for receiving endovascular treatment in accordance with the Chinese Guidelines for Early Endovascular Intervention in Acute Ischemic Stroke 2018 and have successful revascularization (TICI ≥ grade 2b); Subjects or their legal representatives agree to the treatment and sign the informed consent form. Exclusion Criteria: Patients with combined posterior circulation infarction; The mRS ≥ 2 points before the current episode; Patients who are to be treated with or have been treated with anticoagulants; Patients with existing or active organ bleeding within 6 months of enrollment, including cerebral hemorrhage, subarachnoid hemorrhage, gastrointestinal tract hemorrhage, fundus hemorrhage, etc; The presence of other intracranial pathologies, such as cerebrovascular malformations, cerebral venous lesions, tumors, and other diseases involving the cranium; Severe organ dysfunction or failure; Those with severe hematologic disorders or severe coagulation abnormalities； Those with a history of severe trauma or major surgical procedures within 6 months prior to enrollment; Pregnant or lactating women; Patients with a life expectancy of less than 3 months or who for other reasons are unable to complete the study; Unwillingness to be followed up or poor compliance with treatment; Other conditions that the investigator considers unsuitable for enrollment.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 79 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Acute ischemic stroke patients<br>Acute ischemic stroke patients with large vessel occlusion who received mechanical thrombectomy therapy and are successfully revascularized | Diagnostic Test: enzyme-linked immunosorbent assay(ELISA)<br>* After allowing to stand at room temperature for 2h, blood samples are centrifuged at 4 ℃ for 15 min (12000 rpm) to collect the supernatant. Then, the levels of DKK2, IL-6, IL-1β, TNF-α, and IL-10 in the supernatants are tested by ELISA detection.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 90-day Functional Outcome. | The favorable prognosis (modified Rankin score [mRS] score ≤ 2) and the unfavorable prognosis group ([mRS] score ≥ 3) within 90 d after onset. | The mRS score will follow up at 90 days. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Incidence of neurological deterioration | Significantly improved NIHSS score (an increase in the NIHSS score by ≥4 points) within 7 days after onset. | The NIHSS score will evaluat at the point before revascularization treatment, 0 (immediately after revascularization treatment), 24, 48, 72 hours, and 7 days after revascularization treatment. | 
| Incidence of substantial hemorrhagic transformation | Cranial CT examination will perform to detect substantial hemorrhagic transformation (ECASS typing PH2 type) within 72 hours. | At the point before revascularization treatment, 24, and 72 hours after revascularization treatment. | 
| The severity of cerebral edema. | Cranial CT examination will perform to detect the varying severity of cerebral edema groups (divided into 3 groups according to CED scores 1, 2 and 3) within 72 hours. | At the point before revascularization treatment, 24, and 72 hours after revascularization treatment. | 
| Incidence of midline shift ≥ 6mm. | Cranial CT examination will perform to detect the severity of cerebral edema and cerebral hernia. | At the point before revascularization treatment, 24, and 72 hours after revascularization treatment. | 
| Levels of major inflammatory indicators in peripheral blood. | Blood-brain barrier injury-related indicators (MMP-9, ICAM-1) and inflammation-related indicators (IL-6, IL-1β, TNF-α, IL-10). | Before revascularization treatment, 24, 48, 72 hours and 7 days after revascularization treatment. |