Middle Cerebral Artery Occlusion (MCAO)
Objective: Induce cerebral ischemic injury and measure infarct size in the postischemic brain using Middle Cerebral Artery Occlusion (MCAO) model
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Equipment1
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Materials5
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Protocol Steps
MCAO Surgery
Perform Middle Cerebral Artery Occlusion to induce cerebral ischemic injury in the brain
Note: This creates the ischemic core with excitotoxicity-induced acute neuronal death
View evidence from paper
“Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core”
Monitor HMGB1 Release
Assess massive release of HMGB1 into extracellular space immediately after ischemic insult
Note: HMGB1 is a nonhistone DNA-binding protein released as a result of ischemic injury
View evidence from paper
“High-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult”
HMGB1 Downregulation
Apply short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain
Note: This intervention suppresses infarct size, microglia activation, and proinflammatory marker induction
View evidence from paper
“Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction”
Measure Infarct Size
Quantify the size of the ischemic infarct in the postischemic brain tissue
Note: Infarct size is a primary outcome measure of MCAO severity
View evidence from paper
“Measure infarct size in the postischemic brain”
Assess Microglia Activation
Evaluate microglia activation as a marker of brain inflammation in the postischemic brain
Note: Microglia activation is the hallmark of brain inflammation
View evidence from paper
“Supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation”
Measure Proinflammatory Markers
Quantify proinflammatory marker induction in the postischemic brain tissue
Note: Proinflammatory markers indicate the extent of neuroinflammation
View evidence from paper
“Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction”
In Vitro Verification with Primary Cortical Cultures
Treat primary cortical cultures with NMDA to induce excitotoxicity and measure HMGB1 accumulation in culture media
Note: This verifies the proinflammatory cytokine-like function of extracellular HMGB1
View evidence from paper
“HMGB1 was found to accumulate in NMDA-treated primary cortical culture media”
Microglial Activation Assay
Collect supernatants from NMDA-treated cortical cultures and apply to microglial cultures to assess activation
Note: Supernatants trigger microglia activation through HMGB1-dependent mechanisms
View evidence from paper
“Supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation”
Recombinant HMGB1 Treatment
Treat microglial cultures with recombinant HMGB1 to directly assess its role in microglial activation
Note: Direct treatment confirms HMGB1's essential role in microglial activation
View evidence from paper
“Treatment with recombinant HMGB1 also induced microglial activation”
HMGB1 Depletion Control
Treat culture supernatants with anti-HMGB1 antibody or use HMGB1 shRNA expression to deplete HMGB1
Note: HMGB1-depleted supernatants do not trigger microglial activation, confirming HMGB1's essential role
View evidence from paper
“HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not trigger microglial activation”