3.1 Lead induced obviously the learning and cognitive deficits.
By evaluating the spatial learning and memory performance using the Morris water maze test, it was found that the escape latency in lead exposure group was significantly longer at the third and the fourth day (P < 0.001, Fig. 1A). Further analysis found that the escape latency in the high-dose lead exposure group was longer compare with that of the low-dose lead group (P < 0.01, Fig. 1A). This data indicated that lead exposure was associated with decreased learning and memory ability in rats. In space exploration test, the average frequency of crossing the platform in lead exposure groups rats showed a decrease trend (P < 0.001, Fig. 1B) while the rats in high does lead group had the lowest frequency of crossing the platform. However, there was no statistical significance between the low and the high does lead groups. These results demonstrated that lead could gradually impair spatial orientation of rats. With higher concentration of lead exposure, there were signs of more severe injury to these rats’ cognitive function.
3.2 Metal elements change in hippocampus of rats following lead exposure for 3, 6, 9, 12 weeks
We first used ICP-MS to detect the levels of metal elements in the hippocampus of rats. It was found that some divalent metal elements contents were so rare that they can be neglected. The nine divalent metal elements concentration changes in rat hippocampus were obtained for analysis. PCA was applied to extract the 9 elements and their changes in hippocampus after lead exposure. After 3 weeks of lead exposure, six main components were extracted from the hippocampus. Among them, factor 1(F1) included Ca, Cu, Ni, Mg, Fe and Zn accounting for 79.55% of the total variance. Three main components were extracted among lead and control group after 6 weeks lead exposure of which F1 included Ni, Fe and Cu accounting for 71.33% of the total variance. Three main components were extracted from the hippocampus of rats with 9 weeks lead exposure and F1 included Cu, Mg and Ca accounting for 79.39% of the total variance. Meanwhile, four principal components were extracted from all the elements of the rats’ hippocampus after 12-week lead exposure and F1 included Ca, Fe, Zn, Mg and Cu accounting for 85.02% of the total variance. After 3–12 weeks of lead exposure, the principal metallic trace elements components analysis in rats’ hippocampus indicated copper (Cu) detection. The copper concentration in the hippocampus showed significant changes as exhibited in Fig. 2.
3.3 Metal elements change in CSF of rats following lead exposure for 3, 6, 9, 12 weeks
By using the same metal measurement method as analyzing hippocampus, twelve kinds of divalent metals contents in CSF were identified. After 3 weeks of lead exposure, three main components of which in CSF were extracted. F1 included V, Cu and Fe accounting for 78.08% of the total variance. Five main components were extracted after 6 weeks lead exposure, F1 including Fe, Ni, V, Mo, Ca and Cu accounting for 85.36% of the total variance. After 9 weeks of lead exposure, five main components were extracted of which F1 including Cu, V, Ni, Cr, Mg and Ca accounting for 82.77% of the total variance. Five main components were extracted after 12 weeks of lead exposure that F1 including Ni, V, Cu, Fe and Mn accounting for 79.79% of the total variance. F1 extracted from the different exposure duration included V and Cu, and the differences are shown in Fig. 3.
These data suggested that there was significant change of copper level in the hippocampus and CSF of rats at each exposure duration, indicative that lead exposure might result in the copper disorder of rats’ brain.
3.4 The accumulation of copper and lead in plasma and choroid plexus at different lead exposure duration
Our data indicated that lead exposure can affect copper level in CSF and hippocampus, which led us to try to elucidate its mechanism. Therefore, the copper and lead contents from plasma and choroid plexus were studied. The results were shown in Fig. 4. At every 3 weeks after lead exposure, both lead and copper levels in the plasma and choroid plexus were measured. These results showed that the lead and copper levels in the plasma increased after 3 to 12 weeks of lead exposure, conveying that there were increased lead and copper concentration in the peripheral blood after lead exposure.
As we know BCB is barrier regulating ion homeostasis between the brain and the blood, and the choroid plexus is an important component of the BCB. It can be found that at each point of lead exposure the levels of copper and lead were much higher than that in control. Copper and lead gradually accumulate in the choroid plexus during lead exposure for 3 to 12 weeks.
The lead and copper contents in plasma during lead exposure for 3 to 12 weeks were increased compared to the control group. These results predicted that the choroid plexus surface facing the peripheral blood would be at risk for contacting higher concentration of copper and lead. This changed environment may lead to the accumulation of lead and copper in the choroid plexus. Since there were increased lead and copper contents in the choroid plexus, they could affect the function of the choroid plexus. (for plasma n = 12 for choroid plexus n = 12 per group, one-way ANOVA for each group, P < 0.05 for overall acquisition, probe and reversal). In all panels, error bars represent mean ± SD. *P < 0.01 (lead group vs control group)
3.6 Lead exposure resulted in the change of copper transporter expression in choroid plexus.
As shown in Fig. 6, CTR1 and ATP7A were distinctly expressed in the choroid plexus. In the control group, CTR1 was mainly expressed in the cytoplasm and cell membrane. The fluorescence intensity of CTR1 increased in the choroid plexus after lead exposure and primarily concentrated close to the apical border with a small portion along the basolateral membrane in the choroidal epithelial cells. In the control group, ATP7A was mainly expressed in the cytoplasm. The expression of ATP7A was significantly higher in the cytoplasm of choroid plexus with lead exposure than that in the control group. The intracellular ATP7A fluorescence intensity in high does lead group was higher than that in low does lead group. The plasma lead concentration increment correlated positively with the fluorescence intensity of CTR1 and ATP7A
Besides localization of copper transporter in choroid plexus by immunofluorescence confocal microscopy, we also tested the mRNA expressions of ctr1 and atp7a. As was shown in Fig. 7, the mRNA expression of ctr1 in the choroid plexus of low and high lead exposure group was upregulated by 2.69 ± 0.83 and 3.27 ± 0.82 folds respectively (P < 0.05). The atp7a mRNA expression were also significantly upregulated by 1.53 ± 0.33 and 1.91 ± 0.57 fold respectively (P < 0.05). However, there was no significantly changes of copper transporters expression between the low and high dose lead groups. Lead exposure may induce upregulation of ctr1, causing the excess copper absorption into the cell. Excess intracellular copper may cause stress response in the neurons in the brain. The excess intracellular copper might trigger the neuronal attempt to transport copper out of the cell, as evidenced by upregulation of atp7a expression.
3.7 Lead exposure resulted in choroid plexus morphology changes.
As were shown in Fig. 8, transmission electron microscopy was used to observe the cellular structure morphological alteration. Compared with the control group, the nucleus borders of the choroid plexus cells in the lead exposure group were blurred. The morphological structure of nuclear was abnormal with misaligned and diminished microvilli. The mitochondrial morphological structure was abnormal with fused ridges in some of the mitochondria and swollen in others. However, the ultra-structure changes were similar in the choroid plexus epithelial cells between the two lead exposure groups.
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Nuclear
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Microvilli
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Mitochondria
Figure 8 The morphological changes of choroid plexus in lead group and control rats were observed by TEM. The cell nucleus image contained numerous visible cytoplasmic organelles such as mitochondrion (scale bars 2 µm) (Fig. 8 nucleus). The images with the blurred nucleus membrane observed in the figure belong to the lead groups. Surface of control epithelial cells are replete with microvilli. The microvilli in lead group cells were diminished and misaligned (scale bars 1 µm) (Fig. 8 Microvilli). Mitochondria in the control cells showed clear borders and internal ridges. However, the mitochondrial structure of the lead groups demonstrated partial ridge melting. (scale bars 1 µm) (Fig. 8 Mitochondria). (n = 4 per group)