CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat.
Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.
Krupinski
J
J
Unitat de Neuropatologia, Departament de Biologia Cellular i Anatomia Patològica, Universitat de Barcelona, Barcelona, Spain.
Ferrer
I
I
Barrachina
M
M
Secades
J J
JJ
Mercadal
J
J
Lozano
R
R
D000818:Animals; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics; D003566:Cytidine Diphosphate Choline / Q000494:pharmacology* / Q000627:therapeutic use; D053938:DNA Fragmentation / Q000187:drug effects* / Q000502:physiology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D005260:Female; D006868:Hydrolysis; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000201:enzymology*; D007274:Injections, Intraperitoneal; D018697:Nootropic Agents / Q000494:pharmacology / Q000627:therapeutic use; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity / Q000187:drug effects
D061945:Caspase Inhibitors; D004792:Enzyme Precursors; D018697:Nootropic Agents; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D003566:Cytidine Diphosphate Choline; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases