INABIS Y2K, Poster #93, Matsuoka et al. Methods

MATERIALS AND METHODS
Animals:
Male Wistar rats (Crj: Wistar, Charles River, Atsugi, Japan) weighing approximately 300 g were used.

Materials:
-8-Cyclopenthyltheophylline (CPT) and N6-cyclopenthyladenosine (CHA) (A1 adenosine receptor antagonist and agonist, respectively): RBI
-KA: Sigma.
-Mouse monoclonal antibodies against rat MAP-2 and porcine GFAP were from Chemicon International, and rat CD11b and rat MHC class II were from Harlan Sera-Lab.
-Rabbit polyclonal antibodies against c-Jun and Ser73-phosphorylated c-Jun were from New England BioLabs. This anti-c-Jun antibody was raised using a synthetic peptide corresponding to amino acids 1-79 of human c-Jun. The anti-phosphorylated c-Jun antibody was raised using a synthetic phospho-Ser73 peptide corresponding to amino acids 68-77 (LLKLAS*ELERC) of human c-Jun (asterisk indicates phosphorylation). This anti-phosphorylated c-Jun antibody detects phosphorylated c-Jun at Ser73, yet will not react with nonphosphorylated c-Jun.

Drug administration, animal model, and experimental protocol
-CPT was injected i.p. 60 min before KA injection.
-CHA was co-injected with KA.
-One microgram KA was injected into the right lateral ventricle.
-As a control, rats were injected with the same amount of vehicle.
-Each group consisted of five rats.

Immunohistochemistry
-Twelve hours or 4 days after treatment, the animals were perfused with phosphate-buffered saline, followed by a cold fixative consisting of 4% paraformaldehyde in phosphate buffer.
-After perfusion, the brain was quickly removed and post-fixed for 24 hours.
-The free floating sections were incubated with antibodies against MAP-2 (1:3,000), c-Jun (1:200), phospho-c-Jun (1:200), CD11b (1:6,000), GFAP (1:5,000) or MHC class II (1:2,000) for 4 days at 4C.
-The antibody was detected by an ABC Elite kit.

Quantitative analysis and statistical evaluation
-Immunostained sections (2.56 mm caudal from the bregma) were scanned using a camera (KY-F55MD, Victor, Tokyo, Japan) and then analyzed (WinRoof, Mitani Corp., Fukui, Japan).
-For the quantitative analysis of neurodegeneration, the percent of the MAP-2-immunoreactive area of each subfield was measured and used as an index of neuronal survival (decrement indicates neuronal cell loss). The percent of MAP-2-immunoreactive area was calculated by following formula: (MAP-2-immunoreactive area [%]) = (MAP-2-immunoreactive area [mm2]) / (area of subfield, i.e., CA1 or CA3 [mm2]).
-The number of c-Jun- and phosphorylated-c-Jun-immunoporeactive cells was counted in each subfield, i.e., CA1 or CA3, and presented as the number of immunopositive cells in each milisquare [cells/mm2].
-Data are presented as the mean +/- standard error of five rats. The significance of differences was determined by an analysis of variance (ANOVA) with the Bonferroni/Dunn posthoc test (Stat View, Abacus Concepts, Berkeley, CA).