Follistatin-Like 1 Attenuates Apoptosis via Disco-Interacting Protein 2 Homolog A/Akt Pathway After Middle Cerebral Artery Occlusion in Rats

Abstract

Background and Purpose Follistatin-like 1 (FSTL1), an extracellular glycoprotein, has been reported to decrease apoptosis in ischemic cardiac diseases but its effect in ischemic stroke has not been examined. We hypothesized that recombinant FSTL1 attenuates neuronal apoptosis through its receptor DIP2A and the Akt pathway after middle cerebral artery occlusion (MCAO) in rats. Methods One hundred and forty male Sprague-Dawley rats were subjected to 2 hours of MCAO followed by reperfusion. In a subset of animals, the time course and location of FSTL1 and DIP2A were detected by western blot and immunofluorescence double staining. Another set of animals were intracerebroventricularly given either recombinant FSTL1 1 hour after reperfusion or FSTL1-siRNA 48 hours before reperfusion. Additionally, DIP2A was knockdown by siRNA in some animals. Infarction volume and neurological deficits were measured, and the expression of FSTL1, DIP2A, phosphorylated Akt, cleaved caspase-3 and TUNEL were quantified using Western blot. Results The expression of FSTL1 and DIP2A were increased in neurons and peaked 24 hours after MCAO. Recombinant FSTL1 reduced brain infarction and improved neurologic deficits 24 and 72 hours after MCAO via activation of its receptor DIP2A and downstream phosphorylation of Akt. These effects were reversed by DIP2A-siRNA and FSTL1-siRNA. Conclusion Recombinant FSTL1 decreases neuronal apoptosis and improves neurological deficits through phosphorylation of Akt by activation of its receptor DIP2A after MCAO in rats. Thus, FSTL1 may have potentials as a treatment for ischemic stroke patients.

Correcting for Brain Swelling’s Effects on Infarct Volume Calculation After Middle Cerebral Artery Occlusion in Rats

“…Study 1 examines two different doses of a treatment for reducing infarction [22]. When Lin et al’s algorithm is used for calculating the infarct volumes, the MCAO+Treatment Dose 2 group has a significantly smaller infarct volume than both the MCAO and MCAO+Vehicle groups ( p <0.05 for MCAO+Treatment Dose 2 vs. MCAO and MCAO+Vehicle).…”

“…Our algorithm’s infarct volumes are not statistically different from one another. n =6/group [22]. b Study 2.…”

Tyrosine Phosphorylation of NR2B Contributes to Chronic Migraines via Increased Expression of CGRP in Rats

“…In the chronic migraine group, 20  μ L of IS was infused. Following the IS infusion exposures in the CM-genistein and CM-genistein-CGRP groups, intracerebroventricular (ICV) injection of genistein (100 ng/g, 300 ng/g) or genistein and recombinant CGRP (re-CGRP) was performed with a microsyringe after the last IS stimulation, respectively [19]. For the CM-genistein-vehicle group, an equal amount of vehicle was injected.…”

“…Rats were euthanized at 24 hours after genistein treatment for double immunofluorescence staining, as previously described [19], using a neuronal marker (anti-NeuN) (1 : 100, Millipore, Temecula, CA) and NR2B tyrosine phosphorylation (anti-NR2B-pTyr antibody) (1 : 100, Abcam). Brain sections in the trigeminal nucleus caudalis were incubated with a mixture of NeuN and NR2B-pTyr primary antibodies overnight at 4°C, followed by a mixture of secondary antibodies for 60 minutes at room temperature.…”

“…Proteins of the trigeminal nucleus caudalis were extracted by homogenizing in RIPA lysis buffer (sc-24948). Western blotting was performed as previously described [19]. Equal amounts of protein samples (50  μ g) were subjected to SDS-PAGE gel, electrophoresed, and transferred to a nitrocellulose membrane.…”