CREB activation in the rapid, intermediate, and delayed ischemic preconditioning against hypoxic-ischemia in neonatal rat

Abstract

Ischemic preconditioning (IP) is a defense program in which exposure to sublethal ischemia followed by a period of reperfusion results in subsequent resistance to severe ischemic insults. Very few in vivo IP models have been established for neonatal brain. We examined whether rapid, intermediate, and delayed IP against hypoxic-ischemia (HI) could be induced in neonatal brain, and if so, whether the IP involved phosphorylation of cAMP response element-binding protein (pCREB) after HI. Postnatal day 7 rat pups were subjected to HI at 2 h (2-h IP), 6 h (6-h IP), or 22 h (22-h IP) after IP. We found all three IP groups had significantly reduced neuronal damage and TUNEL-(+) cells 24 h post-HI than no-IP group. Compared with control, the no-IP group had significant decreases of pCREB and mitochondria Bcl-2 levels in the ipsilateral cortex 24 h post-HI. In contrast, the three IP groups had increased pCREB and mitochondria Bcl-2 levels, and significant differences were found between three IP and no-IP groups. The increases of cleavage of caspase-3 and poly (ADP-ribose) polymerase and of cells with nuclear apoptosis inducing factor post-HI in no-IP group were all significantly reduced in three IP groups. The increases of caspase-3 and calpain-mediated proteolysis of a-spectrin post-HI were significantly reduced only in 22-h IP group. Furthermore, all three IP groups had long-term neuroprotection at behavioral and pathological levels compared with no-IP group. In conclusion, IP, rapid, intermediate, or delayed, in neonatal rat brain activates CREB, up-regulates Bcl-2, induces extensive brakes on caspase-dependent and -independent apoptosis after HI, and provides long-term neuroprotection.

Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

“…One hour later, the pups were placed in air-tight 500-mL containers with 8% oxygen flow rate of 3 L/min for 2 hours. 11 Pups with no IP underwent sham operation 24 hours before HI, whereas control pups were sham-operated without HI. The preconditioning experiments were performed by a laboratory technician, whereas the quantitative measurement was performed by the investigators who were blinded to the grouping during the assessment.…”

“…11 We used this in vivo IP model and established in vitro OGD preconditioning models in neuronal and endothelial cells to test the hypotheses that: (1) IP upregulates cIAP1 expression in neurons and vascular endothelial cells; (2) cIAP1 is required for preconditioning-mediated protection against HI in vivo and against Background and Purpose-The neurovascular unit is a major target of hypoxia-ischemia (HI) injury in the neonatal brain.…”

Hypoxic–ischemic retinal injury in rat pups

“…After being blocked by 5% goat serum, the sections were incubated with various primary antibodies overnight followed by secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) (25). The primary antibodies used were glial fibrillary acidic protein (1:200, GFAP, Chemicon, Temecula, CA), ED1 (1: 500, Serotec, Raleigh, NC), and activated caspase-3 (1:200, Cell Signaling, Danvers, MA).…”

“…TUNEL TUNEL was performed using an ApopTag fluorescent direct in situ apoptosis detection kit (Chemicon), and samples were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA) (25). The number of TUNEL(+) cells was counted in an area of 40 × 100 μm at 200× magnification, using a fluorescence microscope (Nikon).…”

“…acquired using a Nikon microscope (Nikon, Tokyo, Japan) (25). To quantify the extent of retinal damage, cells were counted in two nonoverlapping views (250 × 250 μm) within central areas (100 μm from the optic disc) from each section by two independent observers (15).…”