B . Lin et al. Brain Research 888 2001 107 –116
109
2.5. Quantitative western blots 2.6 fold by dextrose administration hyperglycemic–is-
chemic rats, 340666 mg dL; normoglycemic–ischemic In a separate series, relative levels of expression of
group, 133621 mg dl. b-APP were determined by quantitative western blot
analysis. The following experimental groups were studied: 3.2. Summary of light-microscopic histopathology
sham normoglycemic n53 and sham hyperglycemic n53 with 24 h recovery; 12.5 min ischemia 124 h
The light-microscopic neuropathological alterations in recovery normoglycemic n54, 12.5 min ischemia 124 h
normoglycemic and hyperglycemic rats surviving for 1 and recovery hyperglycemic n54; 12.5 min ischemia 13
3 days following a 12.5 min ischemic insult have been day recovery normoglycemic n54; and 12.5 min is-
previously reported and illustrated in detail [16]. To chemia 13 day recovery hyperglycemic n54. Tissue
summarize these findings, brains of hyperglycemic –is- samples from hippocampus |100 mg, thalamus |40
chemic rats contained extensive ischemic neuronal altera- mg, and cortex |500 mg of each brain were isolated and
tions and foci of infarction within neocortex, striatum, and rapidly frozen on dry-ice. Total protein was isolated by
thalamus; and widespread hippocampal damage extending homogenization in buffer composed of 50 mM Tris, pH
beyond the CA1 sector. These changes were already 8.0, 100 mM NaCl, 0.1 SDS, 0.01 mg ml leupeptin, and
apparent at 24 h. Vascular changes observed in the hy- 100 mM phenylmethylsulfonyl fluoride PMSF. Protein
perglycemic group consisted of endothelial thickening, concentration was determined using the Bio-Rad DC
vascular occlusion, prominent peri- and intravascular poly- protein assay system.
morphonuclear and monocytic accumulation, and foci of Protein samples were added to 10 SDS and boiled to
perivascular rarefaction and microinfarction. By contrast, denature them. Twenty five mg of protein per sample was
normoglycemic–ischemic rats showed no injury at 24 h. loaded onto a 12 SDS polyacrylamide stacking gel and
At 3 days, the cerebral neocortex and striatum of nor- electrophoresed in 25 mM Tris, 250 mM glycine pH 8.3,
moglycemic–ischemic animals showed only mild damage, 0.1 SDS at 30 mA. Proteins were transferred by standard
with few or no necrotic neurons; the thalamus was spared electroblotting techniques to a PVDF transfer membrane
from injury; hippocampal damage was confined to the CA1 Polyscreen, NEN Research Products in 25 mM Tris, 192
sector; and no endothelial changes were present. Numbers mM glycine, pH 8.3, and 10 methanol. After confirma-
of ischemic cells in the striatum of hyperglycemic animals tion of transfer by Ponceau S Red staining, the membrane
were increased by 5 fold or more compared to nor- was blocked with 5 milk in 13PBS plus Tween-20 for 2
moglycemic rats, and surviving normal neurons in the h at room temperature.
vulnerable hippocampal CA1 sector of hyperglycemic– Incubation with a monoclonal antibody to the N-termi-
ischemic rats were reduced to one third of the number seen nal portion of b-APP695 clone 22C11, 0.1 mg ml,
in normoglycemic animals [16]. Boehringer Mannheim was performed in the same buffer
at 1:1000 dilution at room temperature for 2 h. Following a 3.3. Immunohistochemical observations
brief wash, incubation with HRP-linked anti-mouse IgG 1:1000 was carried out for 45 min. Detection of signal
3.3.1. Sham-operated group was performed with a Phototope-HRP Western blot de-
In four of six sham brains 3 normoglycemic, 1 hy- tection kit New England BioLabs utilizing LumiGlo
perglycemic studied at 3 days, scattered faintly stained, reagent and was visualized on X-ray film. The film was
apparently extracellular foci of b-APP immunoreactivity then digitized, and densitometric measurements were made
were observed on non-counterstained sections, randomly to quantify signals from each sample. Following stripping
distributed within the middle layers of neocortex. These of the antibody, the blot was re-incubated as above with an
foci, however, lacked both a dark brown appearance and a antibody against actin Sigma Chemicals at 1:5000. The
punctate or granular texture in hematoxylin-counterstained actin signal was used as an internal control for evenness of
sections and were only rarely intraneuronal. In three of loading.
four hyperglycemic sham-operated rats with 24 h survival, widespread thalamic neurons tended to exhibit light brown
cytoplasmic b-APP immunostaining that was not present
3. Results in the other sham-operated groups. This faint staining
clearly differed, however, from that to be described below 3.1. Physiological variables
in hyperglycemic–ischemic brains, and it was not observed in neocortex or hippocampus sites prominently affected by
Mean blood pressure before ischemia averaged 125610 hyperglycemic ischemia see below.
mmHg mean6S.D.; arterial pO was 117622 mmHg;
2
arterial pCO , 4063 mmHg; and arterial pH, 7.4260.02. 3.3.2. Hyperglycemic–ischemic brains — 24 h
2
There were no inter-group differences. Compared to values The striking finding in this group was the presence of
in saline-treated rats, plasma glucose levels were elevated prominent high-density intraneuronal b-APP immuno-
110 B
reactivity involving neurons throughout the neocortex Fig. immunoreactivity was observed within shrunken pyramidal
1C–F, hippocampus Fig. 2, and dorsal and ventrolateral neurons of frontoparietal neocortical layer V Fig. 3
thalamus Fig. 1G,H. In neocortex, robust intraneuronal although not in regions of frank infarction. Fewer num-
b-APP immunostaining consistently involved neurons of bers of neurons were affected than at 24 h, however, and
the cingulate gyrus and of all layers of dorsolateral and neurons of cingulate gyrus were typically uninvolved. All
lateral neocortex; large pyramidal neurons were the most four brains with cortical b-APP immunopositivity also
consistently affected Fig. 1. On hematoxylin-counter- contained prominent b-APP reactivity throughout the
stained sections, b-APP-positive neocortical neurons were, entire pyramidal layer of the hippocampal CA1 sector and
in some cases, shrunken and triangular in shape with subiculum; b-APP-immunopositive neurons were shrunken
altered nuclear staining, suggesting ischemic necrosis. and appeared necrotic. By contrast, the non-necrotic
Other b-APP-positive cortical neurons, however, were not neurons of the CA3 sector were immunonegative. One
shrunken, showed normal nuclear and nucleolar morpholo- brain displayed CA1 necrosis and b-APP immuno-
gy, and appeared to be viable. Adjacent HE-stained positivity on one side, but normal CA1 morphology and
sections confirmed that cortical zones of b-APP-immuno- b-APP immunonegativity on the other. The thalamus
positivity corresponded to foci of extensive neuronal contained b-APP-positive neurons in only two cases; and
necrosis, with shrunken cellular contours, cytoplasmic scattered small cells of the striatum were b-APP-positive
eosinophilia, and nuclear condensation. No b-APP-im- in three cases.
munoreactivity was noted, however, in areas of frank infarction.
3.3.5. Normoglycemic–ischemic brains — 3 days In the hippocampus of 24 h hyperglycemic–ischemic
Weak intraneuronal b-APP immunostaining was ob- rats Fig. 2, b-APP-immunostained sections of all six
served only in occasional neurons of parietal neocortex in brains revealed prominent b-APP-positivity involving all
four of five brains studied at 3 days. No b-APP-reactivity hippocampal sectors and extending into the dentate hilus
was present in thalamus, striatum, or hippocampus. Fig. 2E,F. On HE sections, the hippocampal CA1
No extracellular b-APP immunoreactivity was observed sector of these brains contained extensive bilateral is-
in any ischemic brain. chemic necrosis of pyramidal neurons in two of six brains;
predominantly unilateral alterations in two brains; and an 3.4. Quantitation of neocortical b-APP immunoreactivity
absence of ischemic neuronal changes in the remaining two cases. In all brains of this group, the dorsal and
Quantitative analysis of b-APP-positive neurons in ventrolateral regions of thalamus also contained extensive
parietal neocortex of hyperglycemic–ischemic rats re- zones of markedly b-APP-positive neurons Fig. 1G,H;
vealed 5.9 fold mean elevations at 24 h, and 10.6 fold adjacent HE sections revealed these neurons to have the
elevations at 3 days, compared to counts in animals with classic morphologic features of ischemic necrosis. Five of
normoglycemic ischemia Fig. 4. These differences were six rats at 24 h exhibited mild intraneuronal b-APP
highly significant. reactivity within small cells of the striatum. No b-APP
immunoreactivity was present in the corpus callosum. 3.5. Western analysis
3.3.3. Normoglycemic–ischemic brains — 24 h Western blots in all groups revealed a single consistent
The six animals of this group showed only low-density band data not shown. By quantitative densitometry, the
intraneuronal b-APP immunostaining of larger pyramidal overall tissue levels of b-APP in cortex, hippocampus, and
neurons within the dorsolateral and lateral regions of thalamus
were similar
in normoglycemic-
and hy-
frontoparietal neocortex Fig. 1A,B. On hematoxylin- perglycemic–ischemic rats and in sham animals. These
counterstained sections, this b-APP immunopositivity in- data, normalized to actin, are shown in Table 1 for
volved the cytoplasm but not the nuclear region of neurons hippocampus and thalamus. Actin-normalization was not
and was punctate or granular in texture. b-APP-positive available for cortical samples, but no intergroup differ-
neurons appeared morphologically normal, with intact ences in background-corrected optical densities for b-APP
nuclear morphology, visible nucleolus and no cellular were evident.
shrinkage. The cingulate gyrus and thalamus contained no b-APP immunopositivity. The CA1 sector of hippocampus
showed faint b-APP immunoreaction within normal ap-
4. Discussion
