Animal bioassay experiments

Name	Type	Comments
90-day study	Subchronic (30-90 days)	Forty healthy, 5-week old, male Wistar rats (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 90--120 g were housed in an environment of constant temperature with 12 h light/dark cycle and provided with free access to food and water. All animal experiments were approved by the local Institutional Animal Care and Use Committee. All the rats were allowed to adapt for 7 days after arrival to the animal room and were then randomly assigned into four groups: control (distilled water, n=10), fluoride (50 mg/L F-, n=10), aluminum (100 mg/L Al3+, n=10), and combined (50 mg/L F- and 100 mg/L Al3+, n=10). Water containing 50 mg/L F- and 100 mg/L Al3+ was prepared using NaF and AlCl3. After 3 months, the pigment and abrasion of the rats teeth were observed by the naked eye and photographed with a digital camera. The short-term memory ability and learning and memory ability of the rats were examined using Y maze and Morris water maze, respectively. With the completion of Morris water maze, 24 h urine was collected. Then, the rats were sacrificed under chloral hydrate anesthesia. The brain and hippocampus were collected, part of these tissues was used to determine the levels of fluoride and aluminum, part was fixed in 10% formaldehyde solution for HE staining and immunohitochemistry, and part was fixed in 0.25% glutaraldehyde solution at 4 degrees C for transmission electron microscopy.