Central Nervous System Toxicity Of Single-walled Carbon Nanotube In Mice

[Objective]Nanomaterial (NM) has unique physio-chemical properties because of the small size, and was produced for various applications. Carbon nanotube (CNT) are one of the remarkable NM of the world, including Single-walled carbon nanotube (SWCNT) and Muti-walled carbon nanotube (MWCNT). SWCNT has applications in space structures, thermal control, sensing technology, energy, electronic,biomedicine and pharmacy owing to the specific properties.The world widely use of NM (including SWCNT) generated enormous economic and scientific benefits, but at the same time, owing to the world widely utility and exposure, people pay close attention to the assessment of the potential impact on human health and environment suffers in recent years.Studies indicated that the concentration of the nanopartical in the air has the positive relationship to the respiratory and cardiovascular disease. Recent studies have already shown that SWCNT can decrease cell viability, induce apoptosis and change of cell cycles, also can get into the body via inhalation, digestive, skin and injection, then achieved the target organs through lymph and blood circulation induce biotoxicity. SWCNT could get into the lung and cause pulmonary inflammation, intersitital fibrosis and granulomas lesions via the pharyngeal aspiration which is the commonly way. Once nanoparticles distributed in the lung, it can transport from the pulmonary to the extra-pulmonary tissues, distributed to heart, liver, brain and so on then induce tissues damage. All these makes us pay more attention to the extra-pulmonary toxicity of SWCNT. Nowadays, it is evidenced that SWCNT can induce aorta mitochondria damage and oxidative stress, also accelerate atherosclerotic of. Recent studied show that the main machine processed of NM is inflammatory reaction and oxidative stress, SWCNT induce the body produce ROS cause oxidative stress, then lead to inflammatory cause tissue damage.Central nervous system is the chief component element of nervous system, the network or loop are gathered with lots of neurone, is the basement of learning, memories, human consciousness, psychological and thinking, meanwhile regeneration capacity of the nervous tissue is extremely limited. The blood brain barrier (BBB) serves as a physical moshysical barrier and strictly filter the extra-substance into and out of the brain in order to maintain CNS homeostasis. Recent studies indicated that CNT can get into CNS via BBB, olfactory bulb and sensory nerve ending then induce CNS damage. Though the research of the neuro-bio reaction is still deficiency, some researches have already indicated that NP can induce neurotoxicity, cause nervous tissue lesion.SWCNT can distributed in the whole body through different ways and induce inflammatory and oxidative stress in the espiratory and cardiovascular system, and can reach the brain via BBB, so it is impretive to know whether SWCNT effects the BBB and CNS and illustrate the machine. According to these, the objective of this study was to investigate the structure change of the BBB and CNS neurotoxicity, such as re-inflammatory cytokine expressions, ROS production, antioxidant enzyme levels and neurology index in different sub-brain, illustrate the relationship of NM and Central nervous system disease.[Materials and methods] 1.Animals Male ICR mice 22±2 g, housed in plastic cages under controlled environmental conditions, temperature 24±0.5℃, humidity 55±5%, and a 12h light/dark cycle was in effect, with food and water available ad libitum, adapted 1 week before experiment.2.Single-walled Carbon NanotubeSolvent:saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times;SWCNT:The 1 mg/ml SWCNT were suspended in saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8 s×15 times, then immediately diluted to 0.5,0.2.0.1 mg/ml. The suspensions used in each experiment were always prepared freshly.3.TreatmentThe experiment contains dose-dependent and time-dependent study.Dose-dependent study, five groups,100 animals, one control (the saline contained 0.1% Tween80) and 4 treated groups (different concentrations,2.5,5,2.5,25 mg/kg), administered 0.5 ml per mouse for continuous 5 days through tail intravenous injection with the observation time points of 7 days.Time-dependent study, eight groups,220animals, control group (the saline contained 0.1% Tween 80) and treated groups (concentrations,12.5mg/kg), administered 0.5ml per mouse for continuous five days through tail intravenous injection with the observation time points of 1,7,14 and 28 days.The animals were divided randomly into 13 groups, including histopathology, antioxidant enzyme acticities and MDA levels, Western Blotting and PCR four examinattion, each 5 murine. Recorded the body weight before the the administration and the time point of the observation. At the post-administration time point, except the histopathology animals were treated with perfusion then make the slides, others were sacrificed directly, cerebellum, striatum, hippocampus and cerebral cortex were divided on the ice bath and kept in 1.5 ml EP tube under-80℃.4.Nanomaterial character SWCNT 2 mg were suspended in 4 ml saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times, then observed with TEM immediately.5. HistopathologyThe brain tissues were collected by brain perfusion in this study. Briefly, the mouse was anesthetized with sodium pentobarbital (70 mg/kg, i.p.), then perfused through heart with 20 ml saline first and 4% paraformaldehyde 30 ml afterwards. Immediately after perfusion, the sample of cerebellum, striatum, hippocampus and cortex were collected and stored in 4% paraformaldehyde at 4℃until the slides were done. The histopathological tests were performed as standard laboratory procedures. After that do the HE staining and IHC staining (ZO-1, Claudin-1, GFAP), the sections were observed and photos were taken by optical microscope.6. Lipid oxidation and antioxidant enzyme acticities detectionThe samples were weighed and homogenized in saline, centrifuged at 4000×g for 10 min at 4℃, then the supernatants were used to measure total protein concentrations (Bradford's method), the content of malondialdehyde (MDA with thiobarbituric acid, TBA method), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).7. Western blotting (Protein expression of neural lesion)The frozen tissues were weighed and collected total protein using RIPA with 1 mmol/L PMSF, and determined the protein content using Bicinchoninic Acid (BCA) Protein Assay. Western Blotting determine GFAP and NOS1 protein levels in brain regions.8. Inflammatory cytokine (qPCR)The total RNA was collected with RNA pro plus (Takara) in liquid nitrogen, then determined the RNA viscosity and purity. Two-steps RT-PCR kits were used to detect the IL-1βand TNF-αmRNA levels.9. Statistical analysis Each experiment was conducted at least three times. Data were represented as mean±standard deviation (SD). Multi-group comparisons were evaluated using one-way-analysis of variance (ANOVA) followed by Least-significant difference (LSD) in post-hoc test for the experiment groups. Statistical probability of P<0.05 was considered significant.Results1. Nanomaterial characterThe TEM result shows that the SWCNT dispersed like tubes (Φ≤100 nm) or agglomeration (100nm≤Φ≤500 nm). And also can found hollowness tubular shape.2. Weight:Both the dose and time dependent research shows that SWCNT treatement inhibit the the weight gain. In the dose-dependent study, the weight increased fewer in the high dose compared to control, and the 25 mg/kg treatment inhibited the weight gain significantly (P<0.05). However in the time-dependent study, the weight gain less in the treated group, but has no sinificant means (P>0.05).3. ImmunohistochemistyHE staining:SWCNT affected the structure of the brain, cells in striatum, hippocampus and cortex disorganized.The test indicated that SWCNT may increase the GFAP protein expression in the earlier time then decreased, but the ZO-1 and Claudin-1 protein expression first decreased then increased in striatum, hippocampus and cerebral cortex except the cerebellum. In the dose-dependent study, it is indicated that, except the cerebellum, GFAP, ZO-1 and Claudin-1 expression increased at the high concentration, especially at 12.5mg/kg. The time-dependent study shows that the GFAP level was higher in the treatment compared with the control at the time point of 7 and 14 days except the cerebellum, however ZO-1 and Claudin-1 levels only decreased in two sub-brain area (striatum and hippocampus).4. anti-oxidant enzyme and lipid peroxidationThe test indicated that SWCNTs effects the anti-oxidant system balance, act as SOD and GSH-Px activity decreased and MDA level heightened in striatum, hippocampus and cerebral cortex. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that anti-oxidant enzyme SOD and GSH-Px activity attenuated and MDA level advanced in the treated group compared to control, and has a dose-dependent relationship in the low concentration, the 12.5 mg/kg was the peak (P<0.05). The time-dependent study shows that the SOD and GSH-Px activity attenuated first then recovered and MDA shows the same result compared with the control at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas (P<0.05). Hower, no obvious alteration was observed in the cerebellum (P>0.05).5. Protein expression of neural lesionIt was indicated that SWCNT upgrade NOS1 and GFAP protein levels in striatum, hippocampus and cerebral cortex, no obvious change was found in cerebellum. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that NOS1 and GFAP protein levels increased, and found a dose-dependent relationship in the low concentration, the 12.5mg/kg was the peak (P＜0.05), and found the same changes in cerebellum, but no difference (P>0.05). The time-dependent study shows that ompared with the control, the NOS1 and GFAP protein levels increased first then attenuated at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas(P<0.05). However, no obvious alteration was observed in the cerebellum (P>0.05).6.Inflammatory cytokineThe test indicated that SWCNT induce inflammation, cytokine such as IL-1βand TNF-αmRNA level increased in the brain. In the dose-dependent study, the TNF-α mRNA level upgraded in striatum and hippocampus with dose-dependent relationships (P＜0.05), obvious lesion was observed in cerebellum and cortex at the 12.5 mg/kg (P<0.05); IL-1βmRNA level increased in cerebellum in low dose,12.5 mg/kg treatment increased in striatum, IL-ip significantly increased SWCNT groups in hippocampus (P＜0.05), with no changes in cortex. The time-dependent study shows that TNF-a mRNA level increased first then decreased in the four sub-brain regions, in cerebellum, striatum, and coetex TNF-a level were the peak at the first day, at the time point of 7 days in n hippocampus; IL-1βmRNA level has no change in cerebellum, increased significantly at the time point of 7 days in striatum and hippocampus.Conclusion1. SWCNT with tail vein injection may decrease the expression of BBB tight junction protein ZO-1 and Claudin-1, disrupt the balance of antioxidant system, promote the expression of inflammatory cytokines and neural lesion,2. SWCNT has different toxicity in each brain region, striatum and hippocampus were more sensitive than cerebellum and cortex,3. Under these conditions, SWCNT demonstrated a dose-dependent and time-dependent relationship damage in finite concentration, morever the damge is reversible,4. In this research, the machanism of SWCNT may the inflammatory reaction and oxidative stress.