ABSTRACT
The molecular mechanisms underlying preconditioning (PC), a powerful endogenous neuroprotective phenomenon, remain to be fully elucidated. Once identified, these endogenous mechanisms could be manipulated for therapeutic gain. We investigated whether lymphocyte cell kinase (Lck), a member of the Src kinases family, mediates PC. We used both in vitro primary cortical neurons and in vivo mouse cerebral focal ischemia models of preconditioning, cellular injury, and neuroprotection. Genetically engineered mice deficient in Lck, gene silencing using siRNA, and pharmacological approaches were used. Cortical neurons preconditioned with sublethal exposure to NMDA or oxygen glucose deprivation (OGD) exhibited enhanced Lck kinase activity, and were resistant to injury on subsequent exposure to lethal levels of NMDA or OGD. Lck gene silencing using siRNA abolished tolerance against both stimuli. Lck-/- mice or neurons isolated from Lck-/- mice did not exhibit PC-induced tolerance. An Lck antagonist administered to wild-type mice significantly attenuated the neuroprotective effect of PC in the mouse focal ischemia model. Using pharmacological and gene silencing strategies, we also showed that PKCε is an upstream regulator of Lck, and Fyn is a downstream target of Lck. We have discovered that Lck plays an essential role in PC in both cellular and animal models of stroke. Our data also show that the PKCε-Lck-Fyn axis is a key mediator of PC. These findings provide new opportunities for stroke therapy development.
Subject(s)
Brain Ischemia/enzymology , Cerebral Cortex/enzymology , Ischemic Preconditioning/methods , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Neuroprotective Agents/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Brain Ischemia/genetics , Cell Death/physiology , Cerebral Cortex/drug effects , Disease Models, Animal , Gene Silencing/physiology , Glucose/deficiency , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Mice , Mice, Knockout , N-Methylaspartate/toxicity , Neurons/enzymology , Oxygen/pharmacology , Primary Cell Culture , Protein Kinase C-epsilon/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Extracranial stenosis (ECS) or intracranial stenosis (ICS) are independent risk factors for stroke after transient ischemic attack (TIA). We examined the association of the age, blood pressure, clinical features, duration of symptoms and diabetes (ABCD2) score, a validated risk prediction model for stroke after TIA, and the presence of ICS or ECS. METHODS: Vascular imaging and ABCD2 scores were obtained in a retrospective cohort of 77 consecutive patients diagnosed with TIA in a single center emergency department. The association between vascular stenosis and ABCD2 scores and how each related to clinical outcome was examined. RESULTS: In all, 30 (39.2%) TIA patients had 37 stenotic lesions; 15 (40.5%) stenotic lesions were ICS and 22 (59.5%) stenotic lesions were ECS. A total of 7 patients (9.5%) had both ECS and ICS lesions. Patients with ABCD2 > 3 were more likely to have ICS (odds ratio [OR] = 6.25, confidence interval [CI] 1.39-32.44, P = .009) and ECS (OR = 5.25, CI = 1.56-17.66, P = .005). Of the 37 stenotic lesions, 21 (56.7%) were symptomatic; 4 (19.2%) of these had an ABCD2 ≤ 3. At 7 days, there were 4 ischemic strokes, 3 had previously demonstrated symptomatic stenotic lesions, and all had ABCD2 scores > 3. CONCLUSIONS: Compared to patients in the low-risk ABCD2 scores, the patients with medium- to high-risk ABCD2 scores are more likely to have symptomatic and asymptomatic vascular stenotic lesions. However, 1 in 5 patients with low-risk ABCD2 score has symptomatic stenotic lesions, indicating ABCD2 score does not identify all patients with symptomatic stenotic lesions.
