Studies On The Molecular Mechanism Of Brain Injury Induced By Drinking Water Fluorosis

Fluoride is an essential trace element for all mammalian species, but meanwhile its excess is known to be toxic both to human and animals, such as affecting development of children's intelligence, impairing learning and memory ability and so on. Research shows that hippocampus is a target site of fluoride, which is closely related to spatial learning and memory ability. Our previous research indicated that fluoride impairs the synaptic ultra microstructure in rat hippocampus, but the concrete mechanism is still unknown. Post synaptic density 95(PSD-95) is one of the main proteins in post synaptic density; though it has no activity, it can connect NMDA receptor and other related molecules to form a signal-complex and combine the excitatory signal-transduction in synaptic level through its different domains. NMDA receptor plays a very important role in forming the synaptic plasticity. Above all, we presumed that changes of PSD-95 expression level in rat hippocampus can effect the clustering of NMDA receptor and then effects learning abilitiy of animals.A variety of mechanisms have been proposed to explain fluoride-induced toxicity, including free-radical theory. It is considered that fluorosis could enhance the lipid peroxidation in vivo, break the balance between oxidation system and antioxidant system, and change the content of superoxide dismutase (SOD) and malondialdehyde (MDA). SOD and MDA content are usually regarded as one of index to lipid peroxidation level, but their specificity and sensitivity are not good enough because of variety reasons. Thus, we chose rat model of chronic fluorosis to explore the effects of different doses of fluoride on spatial learning and memory firstly on macroscopic level, and then detected synaptic membrane fluidity and expression levels of PSD-95 on microcosmic level, to offer some useful clues for further study of fluoride-induced neurotoxicity and provide a scientific basis for early diagnosis and treatment of fluorosis.Methods:192, just weaning male Sprague-Dawley(SD) rats (64±8.8g) were randomly divided into four groups and given 15,30,60 mg/L NaF solution and distilled water respectively for 9 and 18 months. Rats were fed standard diet throughout the experiment and water was given ad libitum. Animal's behaviors were tested every three months, including locomotor activity and exploratory behavior by open field test, learning and memory ability by morris water maze test; animals were thereafter decapitated when exposure was stopped, then evaluated pathological change in hippocampus with HE staining, membrane fluidity with ESR method, and expression level of PSD-95 in hippocampus CA3 region with IHC method.Results:1. Open field test reveal that chronic fluorosis suppressed rat's locomotor activity and exploratory behavior in extraneous environment, such as reduced locomotion and kearing frequency (p<0.05, p<0.01). The results of morris water maze test showed that mean escape latency and escape length of rat drinking fluoride water was higher when compared with control group. It indicated that chronic fluorosis impaired rat's spatial learning and memory ability. In addition, fluoride toxicity is connected with both fluoride concentration and time. Fluoride concen-tration is higher and exposure time is longer, the toxicity of fluoride may be stronger.2. The results of synaptic membrane fluidity showed that, order parameter(S) and rotational correlation time of synaptic membrane which marked by 16-DSA increased as fluoride concentration increased, which means the hippocampus synaptic membrane fluidity decreased in some degree. There was something need to be noticed that rotational correlation time of rat in 9-month old high-fluoride group decreased insteadly, almost decreased to the level of that in low-fluoride group.3. Chronic fluorosis could impair rat brain nerve cells. Results of HE staining showed that, the distribution of rat hippocampus pyramidal cells were sparse, cell outline disappeared. Result of IHC staning showed that, the expression level of protein PSD-95 decreased as fluoride concentration increasing (p<0.01,p<0.05). It is suggested that chronic would impair nerve cell, down-regulated expression of PSD-95 protein and impacted on aggregation of NMDA receptor complex, resulting in learning and memory ability decline finally.4. Brain aging can decrease rat locomoter activity, spatial learning and memory ability, and membrane fluidity. This research showed that group of 18-month old rat escape latency was significant different (p<0.01) when compared with group of 3-month old rat, and this trend intensified with the increase of fluoride concentration. So we can learn from it that animals'learning abilities and memories can be affected by several factors, including senility and environmental factors as chronic fluorosis.Above all, chronic fluorosis can depress rat locomoter activity and impair spatial learning and memory ability. And excessive fluoride can pass blood-brain barrier, attack the nervous cells membrane, decrease rat hippocampus synaptic membrane fluidity and expression level of post synaptic density protein PSD-95. According to free-radical theory, oxidative stress may be a resource of fluoride toxicity. Since the expression level of PSD-95 is sensitive to chronic fluorosis, we could make further research of fluoride effect in early development stage, and it can offer us a theoretical basis in research of PSD-95 as an environmental neurotoxic substance biomarker.