N .Y. Calingasan, G.E. Gibson Brain Research 885 2000 62 –69
63
concentration of reactive oxygen species in the thalamus 0.1 ml of saline 10 g body weight; Sigma, St. Louis, MO.
[22]. Thus, although the mechanism underlying the region- Dietary restricted controls DR C; n55 received a
specific neurodegeneration during TD is unknown, evi- thiamine-supplemented diet and intraperitoneal saline in-
dence for the role of oxidative stress is mounting. jection 0.1 ml 10 g body weight. The thiamine-deficient
Dietary restriction DR is a well-established means of and thiamine-supplemented diets for the DR TD and DR
prolonging the lifespan of mammals [3,34]. DR may act by C groups, respectively, were fed at 60 of the ad libitum
modulating or reducing oxidative stress in several organ intake.
systems [20]. This hypothesis is further strengthened by For the ad libitum group, TD was induced in 5 animals
recent investigations suggesting that DR increases the AL TD as in the DR group except that the thiamine-
resistance of neurons to a variety of oxidative insults. For deficient diet was not restricted. Ad libitum controls AL
example, DR protects neurons against MPTP-induced C; n55 received an unlimited amount of thiamine-sup-
toxicity [14], excitotoxicity, and metabolic injury [4]. DR plemented diet, and intraperitoneal saline injection.
also protects hippocampal neurons in mice against the deleterious presenilin-1 mutation that is linked to early
2.3. Tissue preparation and immunocytochemistry onset of Alzheimer’s disease [36].
If increased oxidative damage is a key underlying Mice were euthanized with halothane after 11 days of
mechanism for the sensitivity of selective neurons and the TD, and perfused transcardially with 0.9 NaCl solution
blood–brain barrier to TD, then it would be predicted that followed by 4 paraformaldehyde in 0.1 M sodium
the dietary restricted, thiamine-deficient mice would be phosphate buffer PB; pH 7.4. Brains were removed and
less sensitive than the ad libitum fed, thiamine-deficient sectioned 35 mm thick.
group. The current studies tested whether DR affords Free-floating sections to be analyzed for neuronal loss
protection against selective neurodegeneration and blood– were immunostained with an antibody against an excellent
brain barrier breakdown during TD. neuronal marker, neuron-specific nuclear protein, NeuN
[29]. This method eliminates the problem of distinguishing between small interneurons and glial cells, which is
2. Materials and methods encountered in routine hematoxylin-and-eosin or cresyl
violet staining. As in our previous studies [8,10], sections 2.1. Animals
were incubated in 0.05 M potassium phosphate buffered saline KPBS containing 1 NaOH and 3 H O for 30
2 2
Six month-old dietary restricted or ad libitum fed min. After rinsing in KPBS 3 times for 10 min each,
C57BL 6NNia mice were obtained from Harlan Sprague– sections were treated with 0.4 Triton X-100 and 1
Dawley Indianapolis, IN through the National Institute bovine serum albumin BSA in KPBS for 30 min. The
on Aging. C57BL 6NNia mice were used because the TD sections were incubated in NeuN antiserum Chemicon,
pathology in this strain has been extensively studied in our Temecula, CA; 1:1000 in KPBS 1BSA 0.4Triton for
previous reports [7–10], and the DR paradigm has also 18 h. After rinsing in KPBS containing 0.25 BSA and
been characterized in this strain [11]. A stepwise increase 0.02
Triton X-100,
sections were
incubated in
in DR regimen was implemented at the Harlan Sprague– biotinylated anti-mouse IgG 1:200 in KPBS 0.25 BSA
Dawley facility, from 90 of the ad libitum intake at 12 0.02 Triton; Vector Laboratories, Burlingame, CA for 1
weeks of age, 80 at 13 weeks, and 60 at 14 weeks and h, followed by avidin–biotin–peroxidase complex 1:200
beyond. Upon arrival, the animals were housed individual- in KPBS; Vector for 1 h. After rinsing in 0.05 M KPBS,
ly and were maintained under constant temperature 708F, the reaction was developed in 0.05 DAB and 0.003
humidity 50 and 12-h light–dark cycle. The mice were H O in KPBS and stopped with 3 washes of KPBS.
2 2
fed a pelleted diet AIN 76; ICN Nutritional Biochemicals, NeuN-stained sections were also used for the assessment
Cleveland, OH with the dietary restricted group receiving of the blood–brain barrier integrity. Since the monoclonal
60 2.1 g day of the total intake of the ad libitum-fed NeuN antibody requires the use of anti-mouse IgG as
mice. The animals were allowed to acclimatize to the secondary antibody, extravasation of IgG, a measure of
environment for 5 days before induction of TD. All animal blood–brain barrier breakdown [6,32] was immunohistoch-
procedures were approved by the Institutional Animal Care emically detectable in the same sections stained for NeuN.
and Use Committee of the Weill Medical College of The pattern of IgG immunoreactivity in NeuN-stained
Cornell University. sections was identical to that of semiadjacent sections that
were stained specifically for mouse IgG following the 2.2. Induction of thiamine deficiency
standard method for detection of blood–brain barrier disturbances for IgG [32]. IgG immunoreactivity did not
For the DR group, TD was induced in 5 mice desig- interfere with neuronal labeling with NeuN antibody.
nated DR TD which received a thiamine-deficient diet Immunostaining for HO-1 employed a modified avidin–
ICN Nutritional Biochemicals and daily intraperitoneal biotin–peroxidase immunocytochemistry [18] as described
injections of the thiamine antagonist pyrithiamine 5 mg in previously [8,10]. Sections were pretreated with 3 H O
2 2
64 N
in 0.1 M sodium phosphate buffered saline PBS for 30 For each animal, three sections 175 mm apart were
min. The sections were incubated sequentially in a 1 examined. Only the intensely stained cells that were
bovine serum albumin BSA and 0.2 Triton X-100 in distinctly above the background constitutive level of
PBS for 30 min, b HO-1 antibody 1:4000; StressGen staining were counted.
Biotechnologies Corp., British Columbia, Canada in 0.1 M PBS 0.5 BSA for 18 h, c biotinylated anti-rabbit
2.6. Statistical analysis IgG Vector diluted 1:200 in PBS 0.5 BSA for 1 h, and
d avidin–biotin–peroxidase complex Vector diluted All values for neuronal and HO-1-labeled cell counts are
1:200 in PBS for 1 h. The chromogen used was 0.05 expressed as means6standard error of the mean S.E.M..
3,39-diaminobenzidine tetrahydrochloride dihydrate DAB; Statistical significance of group differences was tested by
Sigma containing 0.003 H O in PB. one-way analysis of variance ANOVA followed by the
2 2
The specificity of HO-1 antibody binding was confirmed Bonferroni post hoc test.
by preadsorption experiments. HO-1 antiserum was incu- bated with 20 mg ml purified HO-1 protein StressGen for
3 h at 378C. Adjacent sections from TD brains were
3. Results
