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Impact of early developmental fluoride exposure on the peripheral pain sensitivity in mice
Int. J. Devl Neuroscience 47 (2015) 165–171
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Impact of early developmental fluoride exposure on the peripheral pain sensitivity in mice Jing Ma a,1 , Fei Liu a,b,1 , Peng Liu b , Ying-Ying Dong e , Zheng Chu b , Tie-Zhou Hou a,∗ , Yong-Hui Dang b,c,d,∗∗ a
Affiliated Stomatology Hospital of Xi’an Jiaotong University Health Science Center, Xi’an 710004, Shaanxi, PR China College of Medicine & Forensics, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China c Key Laboratory of the Health Ministry for Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China d Key Laboratory of Environment and Genes Related to Diseases of the Education Ministry, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China e Department of Psychiatry, First Affiliated Hospital of Xi’an Jiaotong, University College of Medicine, Xi’an, Shaanxi, PR China b
a r t i c l e
i n f o
Article history: Received 1 June 2015 Received in revised form 19 September 2015 Accepted 21 September 2015 Keywords: Fluoride Developmental exposure Pain sensitivity BDNF Mice
a b s t r a c t Consumption of high concentration of fluoride in the drinking water would cause the fluorosis and chronic pain. Similar pain syndrome appeared in the patients in fluoride therapy of osteoporotic. The aim of the current study was to examine whether exposing immature mice to fluoride would modify the peripheral pain sensitivity or even cause a pain syndrome. We gave developmental fluoride exposure to mice in different concentration (0 mg/L, 50 mg/L and 100 mg/L) and evaluated their basal pain threshold. Von Frey hair test, hot plate test and formalin test were conducted to examine the mechanical, thermal nociceptive threshold and inflammatory pain, respectively. In addition, the expression of hippocampal brain-derived neurotrophic factor (BDNF) was also evaluated by Western blotting. Hyperalgesia in fluoride exposure mice was exhibited in the Von Frey hair test, hot plate test and formalin test. Meanwhile, the expression of BDNF was significantly higher than that of control group. The results suggest that early developmental fluoride exposure may lower the basal pain threshold and be associated with the increasing of BDNF expression in hippocampus. © 2015 Published by Elsevier Ltd. on behalf of ISDN.
1. Introduction Fluoride is an element that widely exists in the air, rocks, soil and water, and is added to many articles of daily use, such as toothpastes and mouth rinses. However, excessive consumption of fluoride in the drinking water, which is the most common way that people intake fluoride, could cause chronic fluorosis. Epidemiological evidences support that fluoride may induce low back pain in people living in the areas with high levels of fluoride in drinking water for more than 20 years. Namkaew and Wiwatanadate (2012) used a retrospective cohort design
∗ Corresponding author. ∗∗ Corresponding author at: College of Medicine & Forensics, Xi’an Jiaotong University Health Science Center, Yanta Road W. 76#, Xi’an 710061, Shaanxi, PR China. Fax: +86 29 82655116. E-mail addresses: [email protected] (T.-Z. Hou), [email protected] (Y.-H. Dang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.ijdevneu.2015.09.005 0736-5748/© 2015 Published by Elsevier Ltd. on behalf of ISDN.
assessing the dose response of exposure to fluoride-contaminated water to chronic pain in two sub-districts of Chiang Mai, Poo-kha and On-tai, in Thailand. They found that about 348 (65.2%) participants currently had lower back pain; 321 (60.1%) had knee pain, and 198 (37.1%) had leg pain. And this lower back pain was statistically positively related to the average daily fluoride dose of consuming water. Moreover, some researchers also reported the fluoride-specific side effects, the lower-extremity pain syndrome, as the treatment for osteoporosis using the fluoride (Briancon and Meunier, 1981; O’Duffy et al., 1986; Schnitzler and Solomon, 1985). Accumulating evidence has indicated that not only the skeletal organs, but also developing brain is vulnerable to fluoride. Overmuch fluoride intake could damage the integrity of cerebral vascular and neuron (Varner et al., 1998), resulting in the cognition deficiency (Chioca et al., 2008; Niu et al., 2009). Our previous study showed that the fluoride exposure during development affects both cognition and emotion in mice (Liu et al., 2014). The mice presented the anxiety/depression-like behaviors.
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The hippocampus is a component of limbic system and has long been implicated in learning and memory function. Meanwhile, it contributes to the negative affect and approach-avoidance motivation experienced during pain (Duric and McCarson, 2007; McKenna and Melzack, 2001). Microinjection of lidocaine or glutamate receptor antagonists into dorsal hippocampal alleviated formalin-evoked nociceptive behaviors (McKenna and Melzack, 1992, 2001). These mechanisms are not fully understood yet. Previously evidences suggest that some hippocampal neurons respond exclusively to painful stimulation, and after noxious physical stimulation, some anatomical changes occur in dentate gyrus neurons (Dutar et al., 1985; Khanna and Zheng, 1999; Sinclair and Lo, 1986). BDNF meets many criteria to be defined as a neurotransmitter/neuromodulator in nociceptive pathway. In central and peripheral nervous system, BDNF facilitates the survival of certain neuronal population during development. It is also an important modulator of synaptic plasticity (Pezet et al., 2002). Antagonism of BDNF attenuates the second phase of hyperalgesia induced by formalin, indicating that BDNF is involved in some aspects of peripheral inflammation (Kerr et al., 1999; Thompson et al., 1999). BDNF is highly expressed in the limbic system, primarily the amygdala, the hippocampus and the hypothalamus (Malcangio and Lessmann, 2003). The present study examined whether exposing immature mice to fluoride would modify their peripheral pain sensitivity or even cause a pain syndrome. We evaluated the mechanical, thermal nociceptive threshold and inflammatory pain, using the Von Frey hair test, hot plate test (HPT) and formalin test (FT), respectively. In order to explore the underlying mechanism of fluoride-elicited pain response, we evaluated the expression of BDNF in hippocampus. We also monitored the weight of animals and open field test (OFT) was applied to assess the locomotor activity of mice.
2.3. General condition and body weight The general condition of each mouse, including body weight, teeth, skin and hair, mental state, and responsiveness were measured and recorded every week. 2.4. Open filed test (OFT) The OFT was performed as previously reported (Xing et al., 2010). An apparatus consisted of a square box of 45 cm × 45 cm × 45 cm was used to measure the locomotor activity. Mice were placed in the central square of the field at the beginning of the test and were allowed 60 min of free exploration. A video-computerized tracking system (SMART, Panlab SL, Barcelona, Spain) was used to record the distance of traveling as a measure of locomotor activity. 2.5. Von Frey hairs test
2. Materials and methods
Mice were undertaken the Von Frey hairs test from 4-week-old to 8-week-old, 5 times in total. Ten stimuli were made with each of a series of Von Frey hairs (North Coast Medical Inc., Morgan Hill, USA) comprised of the first 11 monofilaments (0.008, 0.02, 0.04, 0.070, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0, and 4.0 g). The test was performed as previously reported by Bourquin et al. (2006). The test was started with filament 0.008 g and if negative responses, the next stiffer monofilament was applied. The monofilament that first evoked a positive response was defined as the threshold and no further monofilaments were applied. The positive response was determined by paw withdrawal occurring twice in the 10 applications. We measured the number of positive withdrawal responses in ascending order for each monofilament of the series. Relative frequency of paw withdrawal, the ratio of positive responses to the total ten times, was calculated as another index to assess the mechanical sensitivity.
2.1. Animals
2.6. Hot plate test (HPT)
Four week-old C57/BL male mice from the Animal Center of the College of Medicine, Xi’an Jiaotong University were housed (26 cm × 18 cm × 13 cm) under a controlled 12-h/12-h light–dark cycle (lights on at 7:00 A.M.) at a room temperature of 22 ± 1 ◦ C and 55 ± 5% humidity. Four mice were housed in each cage. Mice were given free access to water and food. Experiments were performed after 4 weeks of feeding; they were considered adults at 8 weeks. The overall design scheme for behavioral tests is shown in Fig. 1. Before each test, the animals were placed in the laboratory for 30 min to be acclimated to the test environment. The experimental protocols were approved by the Xi’an Jiaotong University Laboratory Animal Administration Committee. All efforts were made to minimize the number of animals used and their suffering.
The HPT was performed after OFT and repeated in the consecutive 3 days at an ascending temperature of 46 ± 1 ◦ C, 50 ± 1 ◦ C, 54 ± 1 ◦ C. The data of each group was averaged in 3 different days. Mice were placed on the hot plate apparatus (RB-200, Tme Technology, Cheng du, China) to assess their thermal nociceptive threshold. The test was a modified version of the experimental
2.2. Fluoride administration Based on fluoride concentrations in drinking water, 24 mice were equally and randomly divided into three groups, that is, control group (distilled water, n = 8), mid fluoride (50 mg/L NaF, n = 8) and high fluoride (100 mg/L NaF, n = 8). There were no significant differences in body weight among groups. During the test, the mice were continually treated with their respective NaF concentrations in drinking water. The blinding method was applied in the experiment, that is, the administration of fluoride, operation of behavior test and the analysis of data were conducted by different people individually.
Fig. 1. Treatment schedules. A total of 24 mice were randomly and equally divided into control group and experimental groups, including mid fluoride and high fluoride. In the period of 28–64 days, mice were continually treated with their respective NaF concentrations in drinking water. Day 28–56: mice were undertaken the Von Frey hairs test every once a week, 5 times in total. Day 58: the locomotor activities of the mice were measured for 60 min in the OFT after the Von Frey hairs test. Day 60–62: the HPT was performed after OFT and repeated in the consecutive 3 days at an ascending temperature of 46 ± 1 ◦ C, 50 ± 1 ◦ C, 54 ± 1 ◦ C. Day 64: mice were received a dorsal surface of the right hind paw subcutaneous injection of 5% formalin with a microinjection. Then the mice were sacrificed and the hippocampus was isolated for Western blotting.
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Table 1 Body weight from 4th to 10th weeks (n = 8 each,¯x ± SEM).
0 mg/L 50 mg/L 100 mg/L
4w
5w
6w
7w
8w
9w
10w
14.3 ± 0.5 13.9 ± 0.6 13.7 ± 0.9
18.6 ± 0.7 18.1 ± 0.7 17.9 ± 0.9
21.4 ± 0.8 20.8 ± 0.6 19.6 ± 0.8
22.1 ± 0.7 22.3 ± 0.5 21.8 ± 0.8
23.3 ± 0.7 23.2 ± 0.4 22.9 ± 0.7
24.0 ± 0.8 23.9 ± 0.4 23.7 ± 0.6
25.8 ± 0.6 25.1 ± 0.4 24.2 ± 0.4
design described previously (Mansikka et al., 2005). The response latency to either a hind-paw lick or jump was recorded. If the animal did not response in 30 s, it was removed from the hot plate avoiding the ambustion, and the latency was recorded as 30 s. 2.7. Formalin test (FT) Before the test, animal was placed in the open Plexiglas box (30 cm × 15 cm × 15 cm) for 30 min to get acclimation. 50 L of 5% formalin was injected subcutaneously into the dorsal surface of the right hind paw with a microinjection. Immediately after the injection, the animal was placed in the same box which permitted observation. The number of spontaneous flinches and the accumulating time of licking the injected paw was calculated every 10 min, for 60 min in all. 2.8. Western blotting (WB) Mice were sacrificed after the behavioral experiments. The hippocampus was isolated on ice. The tissue samples were homogenized in RIPA buffer (1 × phosphate buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% sodium dodecyl sulfate) with protease and phosphatase inhibitor cocktail (Roche, Germany), then protein was extracted with ultrasonic broken method, and centrifuged at 12,000 × g for 5 min at 4 ◦ C. After centrifugation, the supernatant was collected and stored at −80 ◦ C until further use. Protein concentration was determined by the Bradford BCA protein assay. Thirty micrograms of denatured total protein samples were loaded onto 15% SDS-PAGE and then transferred onto a 0.22 m PVDF membrane (Millipore, Bedford, MA, USA) at 0.32 A for 90 min. Then blocking with 5% non-fat milk in ddH2 O for 2 h in room temperature, and subsequently incubating primary antibodies for BDNF (3160-1, 1:1000, rabbit polyclonal, Epitomics)
or -actin (C4, 1:5000, mouse monoclonal, Santa Cruz Biotech) overnight at 4 ◦ C. The following day, we washed membranes in TBST 8 times × 5 min, followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h in room temperature, the secondary antibodies were diluted in blocking buffer with the concentration of 1:3000. After incubation, the membranes rinsed with TBST 5 min × 8 times. Bound antibodies were detected using enhanced chemiluminescence solution (ECL, Pierce Biotechnology Inc., Rockford, IL, USA). Image Lab software (Bio-Rad, USA) was used for quantification. All trials were replicated at least three times. 2.9. Statistical analysis All data were expressed as mean ± standard error of mean (SEM), using SPSS13.0 software for data processing and analysis. One-way analysis of variance (ANOVA) and Dunnett-t post hoc tests were performed to analyze OFT, HPT and WB. Repeated measurement test followed by Dunnett-t post hoc tests were used to analyze body weight changes, Von Frey hair test and FT. P values <0.05 were considered to be statistically significant. 3. Results 3.1. General condition and body weight Mice in the control group exhibited normal activity and fur state. Some fluoride-administered mice showed less movement and wrinkled fur, and fluorosis symptoms on incisor teeth (pale yellow). Body weights in all three groups showed no significant differences before feeding and throughout the study period (Table 1). 3.2. The effect of fluoride on the OFT In the OFT, the distances in the first 10 min were similar among the three groups. The distances of fluoride groups in 1 h were less than those of the control group. This difference; however, was not statistically significant (Fig. 2A and B). 3.3. The effect of fluoride on the Von Frey hairs test
Fig. 2. Open field test. The distances travelled in the first 10 min (A) and 1 h (B) showed no statistically significant differences among the three groups. Each point represents the mean ± SEM of eight mice per group for all experiments.
In the Von Frey hairs test, the withdrawal threshold of the mice in control group did not change obviously. However, the threshold of the fluoride-given mice was on the decline. For the 7 and 8-week-old mice, the withdrawal threshold of the high fluoride group (100 mg/L NaF) was significant lower than that of the control group (p < 0.001). The threshold of the mid fluoride group (50 mg/L NaF) was between the other two groups, but there was no statistically significant difference compared with the control group (Fig. 3A). The relative frequency of paw withdrawal is shown in Fig. 3B–F. The relative frequency of paw withdrawal of the 4-week-old mice showed no difference among the three groups (Fig. 3B). The 5week-old mice in high fluoride group (100 mg/L NaF) were first detected the mechanical pain-like behavior compared with the control group (Fig. 3C). The relative frequency of paw withdrawal of
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Fig. 3. Effect of fluoride on threshold in Von Frey hairs test. (A) The mechanical thresholds of the fluoride-treated mice decreased with continuing fluoride exposure and were significantly reduced compared to those of the control group. (B) The relative frequency of paw withdrawal was similar among the three groups (C–F) at 4 weeks of age, but increased in the fluoride-treated mice between 5 and 8 weeks of age. Data was analyzed using repeated-measures analysis of variance (ANOVA), followed by Dunnett-t post hoc tests. # Denotes p < 0.05, ## denotes p < 0.01 and ### denotes p < 0.001 between the groups. * Denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001 compared to the control group at each time point. Each point represents the mean ± SEM of eight mice per group for all experiments.
the fluoride treatment enhanced as time goes on, which was much more obviously in the 7 and 8-week-old mice (Fig. 3E and F). 3.4. The effect of fluoride on the HPT In the HPT, the temperature-dependent latency was observed in all the three groups, that is, as the temperature rose, the latency was curtated. At the temperature of 46 ◦ C and 54 ◦ C, there were no significant differences of the latency among groups. At the temperature of 50 ◦ C, the latency of the high fluoride group (100 mg/L) was less than that of the control group (p < 0.05) (Fig. 4).
3.5. The effect of fluoride on the FT A subcutaneous injection of formalin resulted in a typical biphasic display of flinches and the accumulating licking time of the injected paw. At the time point of 10 min, 40 min, 50 min and 60 min, the number of flinches in high fluoride group was more than that of the control group, so was the licking time at 10 min (Fig. 5A and B). We divided the 1 h test into two parts, the earlyphase (0–10 min) and the late-phase (20–60 min), and figured up the flinches and licking time respectively by calculating the area under the curve (Fig. 5C and D). The flinches of high fluoride group in both phases were more than that of the control group (Fig. 5C). The licking time of high fluoride group was more than that of the control group only in the early-phase (Fig. 5D). 3.6. The expression level of BDNF in hippocampus As shown in Fig. 6, the expression levels of BDNF appeared to be dose-related with concentration of NaF in drinking water. NaF-treated groups exhibited significantly up-regulated BDNF in hippocampus compared with control group (p < 0.05).
Fig. 4. Effect of fluoride on the latencies of mice in the HPT. At 50 ◦ C, the average latency of the high-fluoride group was less than that of the control group; however, at 46 ◦ C and 54 ◦ C, there were no significant differences among groups. Data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett-t post hoc tests. * Denotes p < 0.05 compared to the control group. Each point represents the mean ± SEM of eight mice per group for all experiments.
4. Discussion In the current study, different concentrations of NaF were added to the drinking water of young mice and changes of peripheral pain sensitivity and hippocampal BDNF expression were investi-
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Fig. 5. Effect of fluoride on flinches and licking times of mice in the FT. (A) The average number of flinches in the high-fluoride group was more than that in the control group at the time points of 10 min, 40 min, 50 min, and 60 min. (B) The average licking time in the high-fluoride group was more than that in the control group at 10 min. These data were analyzed using repeated measures ANOVA, followed by Dunnett-t post hoc tests. ### Denotes p < 0.001 between the groups. * Denotes p < 0.05 and *** denotes p < 0.001 compared to the control group at each time point. (C) For both phases, the areas under the curve of flinches were more for the high-fluoride group than for the control group. (D) The area under the curve of licking time in the high-fluoride group was more than that of the control group only in the early phase. These data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett-t post hoc tests. * Denotes p < 0.05 and ** denotes p < 0.01 compared to the control group at each time point. Each point represents the mean ± SEM of eight mice per group for all experiments.
Fig. 6. Effect of fluoride on the expression of BDNF in hippocampus after the behavioral experiments. (A) The BDNF expression was revealed by Western blotting, and -actin was served as loading control. Shown were representative images. (B) The ratios results showed that fluoride significantly increased expression of BDNF in hippocampus. * Denotes p < 0.05 and ** denotes p < 0.01 compared with the control group (n = 8 for each).
gated. Ingesting fluoride from drinking water is the most common and easily overlooked way of chronic fluorosis. So this protocol represents an adequate model for emulating the effects of fluoride consumption in humans. The major findings were that developmental fluoride exposure leads to the enhanced peripheral pain sensitivity, including the mechanical, thermal and inflammatory hyperalgesia, and the expression of BDNF in hippocampus. Consistent with our previous study, no differences in the growth of body weight and spontaneous activity were found (Liu et al., 2014). The development and locomotor ability of the mice was balanced, which guaranteed the reliability of other nociception tests followed-up.
In Von Frey hairs test and HPT, escape and withdrawal reflex of animals were usually defined as the nociceptive behavior (Yamamoto et al., 2002). Two weeks after administering the fluoride-rich water, the basal mechanical threshold was arisen. As time goes by, the discrepancy between the high-fluoride group and control group become significant. In the HPT, we observed a decrease of withdrawal latencies with the raised temperature. But only at the temperature of 50 ◦ C, the latency of the high fluoride group (100 mg/L) was less than that of the control group. When heated to 54 ◦ C, the differences among groups faded. It is possible that the thermal stimulus was too intense for animals at 54 ◦ C, so all the animals jumped or licked hind-paws quickly to avoid and the differences between groups were eliminated. In addition to the above-mentioned acute pain model, we also used FT as a typical protocol for persistent inflammatory pain. Formalin injection into the rat hind-paw induces agitation behaviors, such as licking, biting and flinching of the injected paw. This process is divided into three phases. The first phase (early-phase) initiates right after the injection and lasts for 5–10 min. It is an acute pain for the injection, on account of the activated C-fiber. The second phase is the rest period and the third one (late-phase) begins from 20 min to 1 h after the injection. The inflammatory pain response caused by formalin is observed in the third phase (Shields et al., 2010). In this study, we observed the flinches and licking time of high fluoride group was more than that of the control group in the early-phase, which was in accordance with the results of mechanical pain in the Von Frey hairs test. The flinches of high fluoride group in the late-phase were also more than that of the control group. Pain during early life usually cause alteration of pain sensitivity (Hermann et al., 2006) and neurodevelopment injury, resulting in disorder in cognition and (or) emotional function (Grunau et al., 2009, 2006). Hippocampus is one of the most robust regulators for pathophysiology of depression (Woolley et al., 1990), and has been concerned with pain-related behaviors for its feasible role in molding affective-motivational response to noxious sensory stimulation (Duric and McCarson, 2007). Researches concerned cognition function in chronic pain model showed possible responsible neural regulators, including BDNF and other neuromodulators were common to pain and cognition (Duric and McCarson, 2006; Moriarty
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et al., 2011). It was found that neurogenesis and expression of BDNF and/or BDNF receptors in hippocampus are decreased in the presence of pain (Ceccanti et al., 2012; Lima et al., 2014). Chronic pain in time of early life damaged short-term memory and decreased levels of BDNF in the hippocampus (Nuseir et al., 2015). The similar results in infant rat also showed that chronic pain would down-regulated BDNF mRNA expression with impairment of spatial learning and memory (Li et al., 2005). Chronic pain might regulate the expression of BDNF through activity-dependent mechanism resulting in biochemical and morphological changes in the existing or generating synapses, which could affect the expression of BDNF and cognition function (Li et al., 2005; Ren, 2007). The expression of BDNF might also be influenced by other factors such as genders and environmental conditions (Chourbaji et al., 2012, 2011, 2008). In the present study, we used only male mice to mitigate the impact of fluctuating sex hormone levels. Other environmental and experimental conditions were exactly the same, such as light–dark cycle, room temperature, humidity, housing and acclimating before tests, which aim to focus on the fluoride-induced pain syndrome. The fluoride-induced pain syndrome has been adverted for decades. Schnitzler and Solomon (1985) treated eight osteoporotic patients with 1.09 mg/kg/d NaF. All of the patients were suffered from the joint pain and swelling in the lower limbs during the therapy. Radiographs taken at 6–8 weeks showed features suggestive of the healing juxta-articular stress micro-fractures in every case. Some researchers deemed that stress micro-fracture was not the only reason for the pain syndrome, but rather a component of the whole process. In Desmond’s study, radiographs revealed stress micro-fractures in only five of the 11 symptomatic patients who were treated with NaF (mean dose 78 mg per day) (O’Duffy et al., 1986). It is supposed that the lower extremity pain syndrome during fluoride therapy was resulted from intense regional bone remodeling, which may be complicated by the stress microfractures. Besides, the excess of fluoride may inhibit the insulin then lead to hyperglycemia, which facilitates the pain syndrome (Courteix et al., 1993). The mechanism of fluoride-induced hyperpathia still remains elusive so far. NaF exposure during early life might induce neurotoxicity, which might damage the existing and generating neurons, and alters behavioral phenotype of mice, including anxiety/depression-like and hyperpathia behaviors. BDNF, as a regulator for neuronal survival, regeneration, outgrowth and overall maintenance (Duman et al., 2000), might play an important role in this procedure. In the present study, the expression of hippocampal BDNF was significantly increased in developmental fluoride exposure mice and seemed to be a dose-response relationship, which is accordance with Jiang’s research in 2014 (Jiang et al., 2014). The up-regulated BDNF may serve to restore neuronal function in brain (Kurauchi et al., 2012; Wu et al., 2008). Observation of ultrastructural changes degenerative neurons in hippocampus detects that accumulational BDNF facilitates proliferation and survival of new neurons (Cotman et al., 2007), which indicated BDNF contributed to the neurons recovery and resisting the neurotoxicity induced by NaF. We speculate that the expression of BDNF may decrease at enough long period of time in case that the neurological damage is hardly to be repaired, which needs further experiment to investigate. In summary, we found developmental fluoride exposure through drinking water (100 mg/L) caused the elevated peripheral pain sensitivity and the pain syndrome, including the mechanical pain, thermal nociceptive threshold and inflammatory pain in mice. The expression of hippocampal BDNF was also up-regulated in the fluoride-treated mice. The potential mechanism of developmental neurotoxicity of NaF might be associated to neurodegenerative changes in brain.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Impact of early developmental fluoride exposure on the peripheral pain sensitivity in mice Jing Ma a,1 , Fei Liu a,b,1 , Peng Liu b , Ying-Ying Dong e , Zheng Chu b , Tie-Zhou Hou a,∗ , Yong-Hui Dang b,c,d,∗∗ a
Affiliated Stomatology Hospital of Xi’an Jiaotong University Health Science Center, Xi’an 710004, Shaanxi, PR China College of Medicine & Forensics, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China c Key Laboratory of the Health Ministry for Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China d Key Laboratory of Environment and Genes Related to Diseases of the Education Ministry, Xi’an Jiaotong University Health Science Center, Xi’an 710061, Shaanxi, PR China e Department of Psychiatry, First Affiliated Hospital of Xi’an Jiaotong, University College of Medicine, Xi’an, Shaanxi, PR China b
a r t i c l e
i n f o
Article history: Received 1 June 2015 Received in revised form 19 September 2015 Accepted 21 September 2015 Keywords: Fluoride Developmental exposure Pain sensitivity BDNF Mice
a b s t r a c t Consumption of high concentration of fluoride in the drinking water would cause the fluorosis and chronic pain. Similar pain syndrome appeared in the patients in fluoride therapy of osteoporotic. The aim of the current study was to examine whether exposing immature mice to fluoride would modify the peripheral pain sensitivity or even cause a pain syndrome. We gave developmental fluoride exposure to mice in different concentration (0 mg/L, 50 mg/L and 100 mg/L) and evaluated their basal pain threshold. Von Frey hair test, hot plate test and formalin test were conducted to examine the mechanical, thermal nociceptive threshold and inflammatory pain, respectively. In addition, the expression of hippocampal brain-derived neurotrophic factor (BDNF) was also evaluated by Western blotting. Hyperalgesia in fluoride exposure mice was exhibited in the Von Frey hair test, hot plate test and formalin test. Meanwhile, the expression of BDNF was significantly higher than that of control group. The results suggest that early developmental fluoride exposure may lower the basal pain threshold and be associated with the increasing of BDNF expression in hippocampus. © 2015 Published by Elsevier Ltd. on behalf of ISDN.
1. Introduction Fluoride is an element that widely exists in the air, rocks, soil and water, and is added to many articles of daily use, such as toothpastes and mouth rinses. However, excessive consumption of fluoride in the drinking water, which is the most common way that people intake fluoride, could cause chronic fluorosis. Epidemiological evidences support that fluoride may induce low back pain in people living in the areas with high levels of fluoride in drinking water for more than 20 years. Namkaew and Wiwatanadate (2012) used a retrospective cohort design
∗ Corresponding author. ∗∗ Corresponding author at: College of Medicine & Forensics, Xi’an Jiaotong University Health Science Center, Yanta Road W. 76#, Xi’an 710061, Shaanxi, PR China. Fax: +86 29 82655116. E-mail addresses: [email protected] (T.-Z. Hou), [email protected] (Y.-H. Dang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.ijdevneu.2015.09.005 0736-5748/© 2015 Published by Elsevier Ltd. on behalf of ISDN.
assessing the dose response of exposure to fluoride-contaminated water to chronic pain in two sub-districts of Chiang Mai, Poo-kha and On-tai, in Thailand. They found that about 348 (65.2%) participants currently had lower back pain; 321 (60.1%) had knee pain, and 198 (37.1%) had leg pain. And this lower back pain was statistically positively related to the average daily fluoride dose of consuming water. Moreover, some researchers also reported the fluoride-specific side effects, the lower-extremity pain syndrome, as the treatment for osteoporosis using the fluoride (Briancon and Meunier, 1981; O’Duffy et al., 1986; Schnitzler and Solomon, 1985). Accumulating evidence has indicated that not only the skeletal organs, but also developing brain is vulnerable to fluoride. Overmuch fluoride intake could damage the integrity of cerebral vascular and neuron (Varner et al., 1998), resulting in the cognition deficiency (Chioca et al., 2008; Niu et al., 2009). Our previous study showed that the fluoride exposure during development affects both cognition and emotion in mice (Liu et al., 2014). The mice presented the anxiety/depression-like behaviors.
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The hippocampus is a component of limbic system and has long been implicated in learning and memory function. Meanwhile, it contributes to the negative affect and approach-avoidance motivation experienced during pain (Duric and McCarson, 2007; McKenna and Melzack, 2001). Microinjection of lidocaine or glutamate receptor antagonists into dorsal hippocampal alleviated formalin-evoked nociceptive behaviors (McKenna and Melzack, 1992, 2001). These mechanisms are not fully understood yet. Previously evidences suggest that some hippocampal neurons respond exclusively to painful stimulation, and after noxious physical stimulation, some anatomical changes occur in dentate gyrus neurons (Dutar et al., 1985; Khanna and Zheng, 1999; Sinclair and Lo, 1986). BDNF meets many criteria to be defined as a neurotransmitter/neuromodulator in nociceptive pathway. In central and peripheral nervous system, BDNF facilitates the survival of certain neuronal population during development. It is also an important modulator of synaptic plasticity (Pezet et al., 2002). Antagonism of BDNF attenuates the second phase of hyperalgesia induced by formalin, indicating that BDNF is involved in some aspects of peripheral inflammation (Kerr et al., 1999; Thompson et al., 1999). BDNF is highly expressed in the limbic system, primarily the amygdala, the hippocampus and the hypothalamus (Malcangio and Lessmann, 2003). The present study examined whether exposing immature mice to fluoride would modify their peripheral pain sensitivity or even cause a pain syndrome. We evaluated the mechanical, thermal nociceptive threshold and inflammatory pain, using the Von Frey hair test, hot plate test (HPT) and formalin test (FT), respectively. In order to explore the underlying mechanism of fluoride-elicited pain response, we evaluated the expression of BDNF in hippocampus. We also monitored the weight of animals and open field test (OFT) was applied to assess the locomotor activity of mice.
2.3. General condition and body weight The general condition of each mouse, including body weight, teeth, skin and hair, mental state, and responsiveness were measured and recorded every week. 2.4. Open filed test (OFT) The OFT was performed as previously reported (Xing et al., 2010). An apparatus consisted of a square box of 45 cm × 45 cm × 45 cm was used to measure the locomotor activity. Mice were placed in the central square of the field at the beginning of the test and were allowed 60 min of free exploration. A video-computerized tracking system (SMART, Panlab SL, Barcelona, Spain) was used to record the distance of traveling as a measure of locomotor activity. 2.5. Von Frey hairs test
2. Materials and methods
Mice were undertaken the Von Frey hairs test from 4-week-old to 8-week-old, 5 times in total. Ten stimuli were made with each of a series of Von Frey hairs (North Coast Medical Inc., Morgan Hill, USA) comprised of the first 11 monofilaments (0.008, 0.02, 0.04, 0.070, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0, and 4.0 g). The test was performed as previously reported by Bourquin et al. (2006). The test was started with filament 0.008 g and if negative responses, the next stiffer monofilament was applied. The monofilament that first evoked a positive response was defined as the threshold and no further monofilaments were applied. The positive response was determined by paw withdrawal occurring twice in the 10 applications. We measured the number of positive withdrawal responses in ascending order for each monofilament of the series. Relative frequency of paw withdrawal, the ratio of positive responses to the total ten times, was calculated as another index to assess the mechanical sensitivity.
2.1. Animals
2.6. Hot plate test (HPT)
Four week-old C57/BL male mice from the Animal Center of the College of Medicine, Xi’an Jiaotong University were housed (26 cm × 18 cm × 13 cm) under a controlled 12-h/12-h light–dark cycle (lights on at 7:00 A.M.) at a room temperature of 22 ± 1 ◦ C and 55 ± 5% humidity. Four mice were housed in each cage. Mice were given free access to water and food. Experiments were performed after 4 weeks of feeding; they were considered adults at 8 weeks. The overall design scheme for behavioral tests is shown in Fig. 1. Before each test, the animals were placed in the laboratory for 30 min to be acclimated to the test environment. The experimental protocols were approved by the Xi’an Jiaotong University Laboratory Animal Administration Committee. All efforts were made to minimize the number of animals used and their suffering.
The HPT was performed after OFT and repeated in the consecutive 3 days at an ascending temperature of 46 ± 1 ◦ C, 50 ± 1 ◦ C, 54 ± 1 ◦ C. The data of each group was averaged in 3 different days. Mice were placed on the hot plate apparatus (RB-200, Tme Technology, Cheng du, China) to assess their thermal nociceptive threshold. The test was a modified version of the experimental
2.2. Fluoride administration Based on fluoride concentrations in drinking water, 24 mice were equally and randomly divided into three groups, that is, control group (distilled water, n = 8), mid fluoride (50 mg/L NaF, n = 8) and high fluoride (100 mg/L NaF, n = 8). There were no significant differences in body weight among groups. During the test, the mice were continually treated with their respective NaF concentrations in drinking water. The blinding method was applied in the experiment, that is, the administration of fluoride, operation of behavior test and the analysis of data were conducted by different people individually.
Fig. 1. Treatment schedules. A total of 24 mice were randomly and equally divided into control group and experimental groups, including mid fluoride and high fluoride. In the period of 28–64 days, mice were continually treated with their respective NaF concentrations in drinking water. Day 28–56: mice were undertaken the Von Frey hairs test every once a week, 5 times in total. Day 58: the locomotor activities of the mice were measured for 60 min in the OFT after the Von Frey hairs test. Day 60–62: the HPT was performed after OFT and repeated in the consecutive 3 days at an ascending temperature of 46 ± 1 ◦ C, 50 ± 1 ◦ C, 54 ± 1 ◦ C. Day 64: mice were received a dorsal surface of the right hind paw subcutaneous injection of 5% formalin with a microinjection. Then the mice were sacrificed and the hippocampus was isolated for Western blotting.
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Table 1 Body weight from 4th to 10th weeks (n = 8 each,¯x ± SEM).
0 mg/L 50 mg/L 100 mg/L
4w
5w
6w
7w
8w
9w
10w
14.3 ± 0.5 13.9 ± 0.6 13.7 ± 0.9
18.6 ± 0.7 18.1 ± 0.7 17.9 ± 0.9
21.4 ± 0.8 20.8 ± 0.6 19.6 ± 0.8
22.1 ± 0.7 22.3 ± 0.5 21.8 ± 0.8
23.3 ± 0.7 23.2 ± 0.4 22.9 ± 0.7
24.0 ± 0.8 23.9 ± 0.4 23.7 ± 0.6
25.8 ± 0.6 25.1 ± 0.4 24.2 ± 0.4
design described previously (Mansikka et al., 2005). The response latency to either a hind-paw lick or jump was recorded. If the animal did not response in 30 s, it was removed from the hot plate avoiding the ambustion, and the latency was recorded as 30 s. 2.7. Formalin test (FT) Before the test, animal was placed in the open Plexiglas box (30 cm × 15 cm × 15 cm) for 30 min to get acclimation. 50 L of 5% formalin was injected subcutaneously into the dorsal surface of the right hind paw with a microinjection. Immediately after the injection, the animal was placed in the same box which permitted observation. The number of spontaneous flinches and the accumulating time of licking the injected paw was calculated every 10 min, for 60 min in all. 2.8. Western blotting (WB) Mice were sacrificed after the behavioral experiments. The hippocampus was isolated on ice. The tissue samples were homogenized in RIPA buffer (1 × phosphate buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% sodium dodecyl sulfate) with protease and phosphatase inhibitor cocktail (Roche, Germany), then protein was extracted with ultrasonic broken method, and centrifuged at 12,000 × g for 5 min at 4 ◦ C. After centrifugation, the supernatant was collected and stored at −80 ◦ C until further use. Protein concentration was determined by the Bradford BCA protein assay. Thirty micrograms of denatured total protein samples were loaded onto 15% SDS-PAGE and then transferred onto a 0.22 m PVDF membrane (Millipore, Bedford, MA, USA) at 0.32 A for 90 min. Then blocking with 5% non-fat milk in ddH2 O for 2 h in room temperature, and subsequently incubating primary antibodies for BDNF (3160-1, 1:1000, rabbit polyclonal, Epitomics)
or -actin (C4, 1:5000, mouse monoclonal, Santa Cruz Biotech) overnight at 4 ◦ C. The following day, we washed membranes in TBST 8 times × 5 min, followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h in room temperature, the secondary antibodies were diluted in blocking buffer with the concentration of 1:3000. After incubation, the membranes rinsed with TBST 5 min × 8 times. Bound antibodies were detected using enhanced chemiluminescence solution (ECL, Pierce Biotechnology Inc., Rockford, IL, USA). Image Lab software (Bio-Rad, USA) was used for quantification. All trials were replicated at least three times. 2.9. Statistical analysis All data were expressed as mean ± standard error of mean (SEM), using SPSS13.0 software for data processing and analysis. One-way analysis of variance (ANOVA) and Dunnett-t post hoc tests were performed to analyze OFT, HPT and WB. Repeated measurement test followed by Dunnett-t post hoc tests were used to analyze body weight changes, Von Frey hair test and FT. P values <0.05 were considered to be statistically significant. 3. Results 3.1. General condition and body weight Mice in the control group exhibited normal activity and fur state. Some fluoride-administered mice showed less movement and wrinkled fur, and fluorosis symptoms on incisor teeth (pale yellow). Body weights in all three groups showed no significant differences before feeding and throughout the study period (Table 1). 3.2. The effect of fluoride on the OFT In the OFT, the distances in the first 10 min were similar among the three groups. The distances of fluoride groups in 1 h were less than those of the control group. This difference; however, was not statistically significant (Fig. 2A and B). 3.3. The effect of fluoride on the Von Frey hairs test
Fig. 2. Open field test. The distances travelled in the first 10 min (A) and 1 h (B) showed no statistically significant differences among the three groups. Each point represents the mean ± SEM of eight mice per group for all experiments.
In the Von Frey hairs test, the withdrawal threshold of the mice in control group did not change obviously. However, the threshold of the fluoride-given mice was on the decline. For the 7 and 8-week-old mice, the withdrawal threshold of the high fluoride group (100 mg/L NaF) was significant lower than that of the control group (p < 0.001). The threshold of the mid fluoride group (50 mg/L NaF) was between the other two groups, but there was no statistically significant difference compared with the control group (Fig. 3A). The relative frequency of paw withdrawal is shown in Fig. 3B–F. The relative frequency of paw withdrawal of the 4-week-old mice showed no difference among the three groups (Fig. 3B). The 5week-old mice in high fluoride group (100 mg/L NaF) were first detected the mechanical pain-like behavior compared with the control group (Fig. 3C). The relative frequency of paw withdrawal of
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Fig. 3. Effect of fluoride on threshold in Von Frey hairs test. (A) The mechanical thresholds of the fluoride-treated mice decreased with continuing fluoride exposure and were significantly reduced compared to those of the control group. (B) The relative frequency of paw withdrawal was similar among the three groups (C–F) at 4 weeks of age, but increased in the fluoride-treated mice between 5 and 8 weeks of age. Data was analyzed using repeated-measures analysis of variance (ANOVA), followed by Dunnett-t post hoc tests. # Denotes p < 0.05, ## denotes p < 0.01 and ### denotes p < 0.001 between the groups. * Denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001 compared to the control group at each time point. Each point represents the mean ± SEM of eight mice per group for all experiments.
the fluoride treatment enhanced as time goes on, which was much more obviously in the 7 and 8-week-old mice (Fig. 3E and F). 3.4. The effect of fluoride on the HPT In the HPT, the temperature-dependent latency was observed in all the three groups, that is, as the temperature rose, the latency was curtated. At the temperature of 46 ◦ C and 54 ◦ C, there were no significant differences of the latency among groups. At the temperature of 50 ◦ C, the latency of the high fluoride group (100 mg/L) was less than that of the control group (p < 0.05) (Fig. 4).
3.5. The effect of fluoride on the FT A subcutaneous injection of formalin resulted in a typical biphasic display of flinches and the accumulating licking time of the injected paw. At the time point of 10 min, 40 min, 50 min and 60 min, the number of flinches in high fluoride group was more than that of the control group, so was the licking time at 10 min (Fig. 5A and B). We divided the 1 h test into two parts, the earlyphase (0–10 min) and the late-phase (20–60 min), and figured up the flinches and licking time respectively by calculating the area under the curve (Fig. 5C and D). The flinches of high fluoride group in both phases were more than that of the control group (Fig. 5C). The licking time of high fluoride group was more than that of the control group only in the early-phase (Fig. 5D). 3.6. The expression level of BDNF in hippocampus As shown in Fig. 6, the expression levels of BDNF appeared to be dose-related with concentration of NaF in drinking water. NaF-treated groups exhibited significantly up-regulated BDNF in hippocampus compared with control group (p < 0.05).
Fig. 4. Effect of fluoride on the latencies of mice in the HPT. At 50 ◦ C, the average latency of the high-fluoride group was less than that of the control group; however, at 46 ◦ C and 54 ◦ C, there were no significant differences among groups. Data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett-t post hoc tests. * Denotes p < 0.05 compared to the control group. Each point represents the mean ± SEM of eight mice per group for all experiments.
4. Discussion In the current study, different concentrations of NaF were added to the drinking water of young mice and changes of peripheral pain sensitivity and hippocampal BDNF expression were investi-
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Fig. 5. Effect of fluoride on flinches and licking times of mice in the FT. (A) The average number of flinches in the high-fluoride group was more than that in the control group at the time points of 10 min, 40 min, 50 min, and 60 min. (B) The average licking time in the high-fluoride group was more than that in the control group at 10 min. These data were analyzed using repeated measures ANOVA, followed by Dunnett-t post hoc tests. ### Denotes p < 0.001 between the groups. * Denotes p < 0.05 and *** denotes p < 0.001 compared to the control group at each time point. (C) For both phases, the areas under the curve of flinches were more for the high-fluoride group than for the control group. (D) The area under the curve of licking time in the high-fluoride group was more than that of the control group only in the early phase. These data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett-t post hoc tests. * Denotes p < 0.05 and ** denotes p < 0.01 compared to the control group at each time point. Each point represents the mean ± SEM of eight mice per group for all experiments.
Fig. 6. Effect of fluoride on the expression of BDNF in hippocampus after the behavioral experiments. (A) The BDNF expression was revealed by Western blotting, and -actin was served as loading control. Shown were representative images. (B) The ratios results showed that fluoride significantly increased expression of BDNF in hippocampus. * Denotes p < 0.05 and ** denotes p < 0.01 compared with the control group (n = 8 for each).
gated. Ingesting fluoride from drinking water is the most common and easily overlooked way of chronic fluorosis. So this protocol represents an adequate model for emulating the effects of fluoride consumption in humans. The major findings were that developmental fluoride exposure leads to the enhanced peripheral pain sensitivity, including the mechanical, thermal and inflammatory hyperalgesia, and the expression of BDNF in hippocampus. Consistent with our previous study, no differences in the growth of body weight and spontaneous activity were found (Liu et al., 2014). The development and locomotor ability of the mice was balanced, which guaranteed the reliability of other nociception tests followed-up.
In Von Frey hairs test and HPT, escape and withdrawal reflex of animals were usually defined as the nociceptive behavior (Yamamoto et al., 2002). Two weeks after administering the fluoride-rich water, the basal mechanical threshold was arisen. As time goes by, the discrepancy between the high-fluoride group and control group become significant. In the HPT, we observed a decrease of withdrawal latencies with the raised temperature. But only at the temperature of 50 ◦ C, the latency of the high fluoride group (100 mg/L) was less than that of the control group. When heated to 54 ◦ C, the differences among groups faded. It is possible that the thermal stimulus was too intense for animals at 54 ◦ C, so all the animals jumped or licked hind-paws quickly to avoid and the differences between groups were eliminated. In addition to the above-mentioned acute pain model, we also used FT as a typical protocol for persistent inflammatory pain. Formalin injection into the rat hind-paw induces agitation behaviors, such as licking, biting and flinching of the injected paw. This process is divided into three phases. The first phase (early-phase) initiates right after the injection and lasts for 5–10 min. It is an acute pain for the injection, on account of the activated C-fiber. The second phase is the rest period and the third one (late-phase) begins from 20 min to 1 h after the injection. The inflammatory pain response caused by formalin is observed in the third phase (Shields et al., 2010). In this study, we observed the flinches and licking time of high fluoride group was more than that of the control group in the early-phase, which was in accordance with the results of mechanical pain in the Von Frey hairs test. The flinches of high fluoride group in the late-phase were also more than that of the control group. Pain during early life usually cause alteration of pain sensitivity (Hermann et al., 2006) and neurodevelopment injury, resulting in disorder in cognition and (or) emotional function (Grunau et al., 2009, 2006). Hippocampus is one of the most robust regulators for pathophysiology of depression (Woolley et al., 1990), and has been concerned with pain-related behaviors for its feasible role in molding affective-motivational response to noxious sensory stimulation (Duric and McCarson, 2007). Researches concerned cognition function in chronic pain model showed possible responsible neural regulators, including BDNF and other neuromodulators were common to pain and cognition (Duric and McCarson, 2006; Moriarty
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et al., 2011). It was found that neurogenesis and expression of BDNF and/or BDNF receptors in hippocampus are decreased in the presence of pain (Ceccanti et al., 2012; Lima et al., 2014). Chronic pain in time of early life damaged short-term memory and decreased levels of BDNF in the hippocampus (Nuseir et al., 2015). The similar results in infant rat also showed that chronic pain would down-regulated BDNF mRNA expression with impairment of spatial learning and memory (Li et al., 2005). Chronic pain might regulate the expression of BDNF through activity-dependent mechanism resulting in biochemical and morphological changes in the existing or generating synapses, which could affect the expression of BDNF and cognition function (Li et al., 2005; Ren, 2007). The expression of BDNF might also be influenced by other factors such as genders and environmental conditions (Chourbaji et al., 2012, 2011, 2008). In the present study, we used only male mice to mitigate the impact of fluctuating sex hormone levels. Other environmental and experimental conditions were exactly the same, such as light–dark cycle, room temperature, humidity, housing and acclimating before tests, which aim to focus on the fluoride-induced pain syndrome. The fluoride-induced pain syndrome has been adverted for decades. Schnitzler and Solomon (1985) treated eight osteoporotic patients with 1.09 mg/kg/d NaF. All of the patients were suffered from the joint pain and swelling in the lower limbs during the therapy. Radiographs taken at 6–8 weeks showed features suggestive of the healing juxta-articular stress micro-fractures in every case. Some researchers deemed that stress micro-fracture was not the only reason for the pain syndrome, but rather a component of the whole process. In Desmond’s study, radiographs revealed stress micro-fractures in only five of the 11 symptomatic patients who were treated with NaF (mean dose 78 mg per day) (O’Duffy et al., 1986). It is supposed that the lower extremity pain syndrome during fluoride therapy was resulted from intense regional bone remodeling, which may be complicated by the stress microfractures. Besides, the excess of fluoride may inhibit the insulin then lead to hyperglycemia, which facilitates the pain syndrome (Courteix et al., 1993). The mechanism of fluoride-induced hyperpathia still remains elusive so far. NaF exposure during early life might induce neurotoxicity, which might damage the existing and generating neurons, and alters behavioral phenotype of mice, including anxiety/depression-like and hyperpathia behaviors. BDNF, as a regulator for neuronal survival, regeneration, outgrowth and overall maintenance (Duman et al., 2000), might play an important role in this procedure. In the present study, the expression of hippocampal BDNF was significantly increased in developmental fluoride exposure mice and seemed to be a dose-response relationship, which is accordance with Jiang’s research in 2014 (Jiang et al., 2014). The up-regulated BDNF may serve to restore neuronal function in brain (Kurauchi et al., 2012; Wu et al., 2008). Observation of ultrastructural changes degenerative neurons in hippocampus detects that accumulational BDNF facilitates proliferation and survival of new neurons (Cotman et al., 2007), which indicated BDNF contributed to the neurons recovery and resisting the neurotoxicity induced by NaF. We speculate that the expression of BDNF may decrease at enough long period of time in case that the neurological damage is hardly to be repaired, which needs further experiment to investigate. In summary, we found developmental fluoride exposure through drinking water (100 mg/L) caused the elevated peripheral pain sensitivity and the pain syndrome, including the mechanical pain, thermal nociceptive threshold and inflammatory pain in mice. The expression of hippocampal BDNF was also up-regulated in the fluoride-treated mice. The potential mechanism of developmental neurotoxicity of NaF might be associated to neurodegenerative changes in brain.
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