Our results showed that acute exposure to diesel exhaust particles (DEPs) may impair performance in acquisition phase of Morris water maze test in mice. Both experiment 1 and 2 in this part of study revealed that mice needed more time and distanced moved to reach the platform in acquisition phase after exposure to DEPs.
Cumulative distance from the center of platform quadrant in the experiment 1 or from the center of platform in the experiment 2 (searching error) was also longer in
acquisition phase after exposure to DEPs. Particularly in the experiment 2 with exclusion of mice with low motivation to swim, mice in total 300 μg DEPs exposure group may significantly require more time to find the platform and make more searching error. However, performance in total 100 μg DEPs exposure group was comparable with control group. These results in the experiment 2 showed a possible dose response on acute DEPs exposure. Distance mice moved in acquisition phase also showed a possible dose response manner which was close to significance.
Histopathological examination found no significant difference between total 300 μg DEPs group, total 100 μg DEPs group and control group.
5.1 DEPs dose and effects in neurofunctions
We observed acquisition impairment in both total 750 μg and 300 μg DEPs exposure group and no clear effects in total 100 μg DEPs exposure group. A total 300 μg DEPs were given to mice in two weeks. This was equivalent to exposure to near 200 μg/m3 DEPs for two weeks. Considering deposition rate of inhaled particles, mass concentration of exposed DEPs maybe even higher. US EPA reviewed that mass
concentration of DEPs in the air was about 1 to 4 μg/m3 in the environmental which was
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a way lower than equivalent mass concentration in current study[10].
DEPs concentration in occupational environment, on the other hand, was higher.
Railroad workers may expose to DEPs with 37 to 191 μg/m3 and firefighters may expose to DEPs with 4 to 478 μg/m3[10]. Equivalent mass concentration using in current study was close to these occupational environment and found behavioral
changes in acquisition. It was reported that railroad works with long-term exposure and electricians with relatively short-term exposure to diesel exhaust may have
neurobehavioral effects[55]. Human volunteers who acute exposure to diesel exhaust with 300 μg/m3 for one hour also showed changes in EEG[61]. Although the exposure route and duration were not exactly the same, both previous and our results showed that exposure to higher DEPs may have effects in neurofunctions.
5.2 DEPs characterization and DEPs effects
Although we didn’t measure the characterization of DEPs suspension using in this study, other study measured DEPs size in a similar condition revealed that DEPs size in suspensions was 402 nm[80]. Small particles with relative higher surface area may attach organic chemicals and metals. A study showed that particle core of DEPs and extractable chemicals of DEPs may have different toxicological effects[81]. Whether DEPs with different sizes and whether particle core or chemical extract of DEPs cause different effects in the CNS required more studies to verify.
Different sources of DEPs may result in different chemical composition. We used SRM 2975 in this study which was generated from forklifts with diesel engines. A study compared the difference between forklift DEPs and automobile DEPs and found
variations in chemical composition. This study also conducted a toxicological
experiment using these two DEPs but found differences in biological effects. Chemical
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composition may play roles in this findings. [82] CNS effects cause by different sources of DEPs were worth further studies.
5.3 CNS effects induced by DEPs
As reviewed in Table 1, most studies used diesel exhaust to evaluate behavioral changes and other CNS effects in mature mice. These studies were conducted in a relative long-term exposure [14-16]. Sub-chronic exposure to diesel exhaust has showed impairment in acquisition using the same behavioral test, Morris water maze[16]. Here, we found that acute exposure to DEPs may cause similar effects in the CNS with changes in acquisition ability.
In the study conducted by Win-Shwe et al, mice were exposed to diesel exhaust with DEPs and gases (whole DE) or filtered diesel exhaust with only gaseous portion of diesel exhaust (F-DE). In acquisition phase of Morris water maze test, they found differences between whole DE and control group, but there were no difference between DE and control. Moreover, they found differences between whole DE group and F-DE group. This showed that particles part rather than gaseous part in diesel exhaust may cause poor performance in acquisition phase of Morris water maze.[16] Our results were consistent with their findings.
Limited studies used the same commercial DEPs (SRM 2975) in our experiment to study CNS effects induced by DEPs. In a study conducted by Hougaard et al., mice were exposed to resuspended DEPs (SRM 2975) 90 minutes/day for four days and found no changes in Morris water maze test[62]. The mass concentration of resuspended DEPs using in that study was 71.5 mg/ m3 which was higher than our study, but there were differences in exposure schedule and exposure route between these two studies. As mentioned by Hougaard et al, volatile and semivolatile chemicals were
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eliminated in resuspended DEPs. With many attached volatile and semivolatile
chemicals, it may be the reason that there were differences between previous study and current study [83].
Whether acute effects we found in this study may recover was unknown under current study design. Recent studies targeted at postnatal exposure to concentrated ambient particles found that early exposure may cause neurobehavioral changes and lateral ventricle dilation in the adulthood [70, 71, 84]. This results indicated that articles exposure in developmental period may have persistent effects in the CNS. In reality, organisms exposed to particles air pollution through their whole life, so whether exposure in adulthood may persist was worth further research.
5.4 Mechanism involved in DEPs induced CNS effects
Possible mechanism involved in DEPs induced changes in acquisition phase of Morris water maze was neuroinflammation. A study found that, twenty hours after 20mg/kg DEPs (SRM 2975) exposure in rats, TNF-α in both serum and the brain was elevated. Microglial expression was also increased. This implicated that DEPs may cause systemic effects and may further induced neuroinflammation.[19] Since the same DEPs were used in previous study and current study, our results may share similar mechanism. Previous studies using diesel exhaust also found that pro-inflammatory markers were elevated in multiple brain regions, including the hippocampus, midbrain, olfactory bulb, frontal lobe, temporal lobe and striatum[16-19]. Microglial activation was also observed in the hippocampus[14]. We should then conduct other tests in pro-inflammatory cytokines and microglial activation in brain regions to support the results of acquisition impairment in this study. Since the hippocampus was the major brain region involved in spatial learning and memory, further studies should put more
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emphasis on this part of the brain.
As for histopathology, we didn’t observed changes in multiple brain regions after DEPs exposure. Studies showed that maternal exposure to diesel exhaust may resulted in apoptosis in the cortex, hippocampus and cerebellum [85, 86]. On the one hand, these studies observed apoptosis after maternal exposure and this exposure was in a relatively vulnerable period. In our study, DEPs were given to mice in adulthood. On the other hand, other studies found that cognitive changes were related to subtle morphological changes in axons or dendrites in the hippocampus [9, 87, 88]. Further analysis in detailed morphological or histopathological examination was required to verify current results in behavioral test.
5.5 Spatial learning and memory
We observed significant results in acquisition phase and non-significant results in probe test in current study. This maybe a result of different regions involved in memory acquisition, memory consolidation and memory recall[38]. A study showed that
hippocampus CA3 region may involve in acquisition phase and memory consolidation but not in memory recall[37]. This implied that, in a complicated process of learning and memory, different regions in the brain and even in the hippocampus was involved with different function. Current study found changes in acquisition phase but not in probe test may due to different regions with different impacts to the exposure.
Or, the current study design with one-day probe trial after acquisition phase may not sensitive enough to show the difference in experimental and control groups. A study conducted Morris water maze comparing the different interval between acquisition phase and probe test. It was found that preference for the quadrant previously with the platform (platform quadrant) was decreased when the interval between acquisition
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phase and probe test increased. They found no significant preference in probe test 24 hours after acquisition phase which interval was used in current study.[89] Although they conducted the experiment using rats and a different procedure for Morris water maze, our results without differences in probe test may share the same reason.
5.6 Limitations
We measured swimming velocity of mice in the Morris water maze to check the swimming ability was the same between groups. Additionally, cued platform training in the Morris water maze test or other physical measurements may provide additional information to make sure that there are no difference in visual acuity and swimming ability between groups. Moreover, mice was more stressful than rats[90]. This may cause a failure in behavioral tests. We found that near half of mice in the experiment 2 swam slower. Stress in the Morris water maze may be the reason. Rats which were less stressful in the Morris water maze may be used to confirm current findings.
Since previous study showed that multiple regions may affect by PM, many other behavioral tests related to different brain regions may conducted to confirm PM induced toxicity. On the other hand, further biochemical examinations like cytokines,
inflammatory cells examinations like microglia and detailed histopathological examination were essential to support our findings.
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