Materials and Methods
Animals and Experimental Design
Forty healthy 6-week-old male Wistar rats were randomly
assigned to four groups of ten animals in each. Control animals
consumed the regular nonfluoridated tap water (~0.4 ppm F
natural level). Rats from the other three groups received the
same water supplemented with 2, 10, or 20 mg F/l (as NaF) for
12 months. The animals were housed in a temperature-
controlled room at 20–23 °C under a 12-h light/dark cycle
with free access to the standard rat chow and water ad libitum.
Animal body weight and amount of water ingested by each
group was monitored monthly and at the end of experiment.
The study was approved by the Institutional Animal Care and
Use Committee in compliance with the Animal Welfare Act
and Guidelines for Use and Care of Laboratory Animals.
The rats were killed by lethal ester anesthesia, and blood
from the abdominal aorta was collected into heparinized
tubes. Aliquots of whole blood were taken for determination
of hematocrit (Hct) and reticulocyte number. Then the blood
was centrifuged (3,000 rpm at 20 °C for 5 min) and plasma
was aspirated for determination of F concentration. Upper
layer of white cells was discarded, and erythrocytes were
washed three times with the same solution. The resulting
erythrocytes were suspended in the standard incubation
medium to 10 % Hct.