Zinc Protects The Change Of The Blood-Brain Barrier Induced By Aluminum In Juvenile Rats

ObjectiveAluminum (Al) neurotoxicologic effects attact our attention increasingly. Studiesshowed Aluminum could damage neurons and affect intelligence development. Centralnervous system dysfunction induced by Aluminum is a long and chronic progress.Recent investigation demonstrates that the primary lesion in Alzheimer's disease anddialysis dementia has been postulated to be an impaired BBB permeability that allowsAl to reach the central nervous system. The accumulation of Aluminum in the brain isknown to cause neurodegenerative disorders and it's important to take effectivemeasures to protect brain from Aluminum accumulation.Aluminum can go through and change the permeability of BBB, Zinc (Zn) canprotect in some degree but the dose of Zinc has not been clarified.In this study, we establish Al-toxie rat model and choose sulfate zinc as a protectorto see if Zinc could prevent or protect the integrity of BBB in juvenile rats that exposedto Al in a short time and low doses.Materials and MethodsMaterials:1. Experimental animals: The 40-day old SD rats (80±10g).2. Experimental reagents: A. P. AlCl₃·6H₂O, A. P. ZnSO₄, EB, G250; ratmonoclonal anti-occludin antibody, ECL chemiluminescence kit; goat anti-rabbitantibody conjugated with horseradish peroxidase and SABC immunohistochemistry kit; phalloidine conjugated with rhodamine.3. Experimental instruments: Morris water maze system, low temperaturerefrigeration centrifuge, ultraviolet spectro photometer, automatic running gel imagingsystem, GP electrophoresis apparatus, upright fluorescence micro scope, deep freezerefrigerator, homeothermia freezing microtome, Motic Images Advanced 3.2 imageanalysis system, JEM-1200 transmission electron microscope.Methods1. Groups:300 SD rats were divided into 10 groups at random: A normal saline; B low-Al; Cmid-Al; D high-Al; E low-Zn; F high-Zn; G mid-Al+low-Zn; H mid-Al+high-Zn;I high-Al+low-Zn; J high-Al+high-Zn.2. Drugs adrnin:Aluminum chloride was intrapedtioneally injected, and sulfate zinc was orallyinfused. Four days as a cycle, 8 cycles in total.3. The weight growth of the rats in each group.4. Morris water amaze system was used to test the learning and memoryability.5. Measurement of BBB permeability by EB.6. The ultrastructure change of BBB was observed with transmissionelectron microscope.7. Fluorescence microscope was used to detect the expression of F-actinin brain capillary endothelium.8. Immunohistochemistry was used to detect the localization and theexpression of occludin in brain capillary endothelium. 9. Expression levels of tight junction protein occludin by western blot.10.All data were analyzed by SPSS 13.0, P＜0.05 was consideredstatistically significant.Results1. The weight growth of the rats in each groupCompared with normal saline group(115.6±4.8g), the weight growth in mid-Algroup(82.4±22.2g), high-Al group (55.8±6.80g) were significantly, P＜0.05; mid-Algroup (82.4±22.2g) vs. mid-Al+low-Zn group (108.8±12.4g), mid-Al+high-Zn group(107.0±9.59g), P＜0.01; high-Al group (55.8±6.80g) vs. high-Al+low-Zn group(93.0±4.47g), high-Al+high-Zn group (105.0±10.0g), P＜0.01.2. Morris water maze trainingHidden platform trials (escape latency) and probe trials were not statisticallysignificant between each group, P＞0.05.3. Measurement of BBB permeability by EBThe content of EB in low-Al group(0.053±0.007μg/g brain weight), mid-Al group(0.085±0.012βg/g brain weight), high-Al group (0.110±0.021μg/g brain weight) vs.normal saline group (0.011±0.003μg/g brain weight), P＜0.01; mid-Al group(0.085±0.012μg/g brain weight) vs. mid-Al+low-Zn group(0.036±0.004μg/g brainweight), mid-Al+high-Zn group (0.031±0.002μg/g brain weight), P＜0.01; high-Al(0.110±0.021μg/g brain weight) vs. high-Al+low-Zn group (0.061±0.003μg/g brainweight), high-Al+high-Zn group (0.042±0.006μg/g brain weight), P＜0.01.4. The ultrastructure of BBB in each groupThe cleavage on cell, aggregation of the heterochromation, mitochondriavacuolization, cristae, the blood capillary lumen was shrinked, tight junction wasopened in Al-toxic groups; A slight cleavage of the capillary endothelium, the electron density were enhanced on tight junction connection, basal membrane was clear inAl-Zn groups.5. The fluorescence expression of F-actin in brain capillary endotheliumUnder fluorescence microscope, the red areas are F-actin localizing along thecapillary endothelial cells. The expression of F-actin on mid-Al and high-Al groups areunclear, unconnected, the lumen of BBB was shrinked; The expression of F-actin onlow-Al group, low-Zn group, mid-Al+high-Zn group were nearly integrated, the redfluorescence were connected, bright, clear.6. Immunohistochemistry was used to detect the localization andexpression of occludin protein in brain capillary endotheliumIn Al-toxic groups, occludin protein express slightly; in saline group, low-Al andAl-Zn group, the occludin expressed mainly on the capillary cell membranes in brownblot or brown line.7. The expression of occludin by western blotCompared with saline group, the expression of occludin in Al-toxic groups weresignificantly decreased; compared with the Al-toxic groups, the expression of occludinin Al-Zn groups were increased.DiscussionBBB separates the brain microenviroument from the systemic circulation andmaintains the homeostasis of the central nervous system. Excessive uptake ofAluminum may disturb the learning and memory of humans or animals, and this is achronic progress, happened in every brain development stage. In our study, we use40-day old juvenile SD rats, metal-toxic influenced 32 days, and this is different fromother studies. The learning and memory ability of the juvenile rats were not changeddistinguishedly, suggested that learning and memory ability changed by Aluminum arerelated to the time and the doze of Aluminum exposure.BBB protects the CNS as a stable circumstance. The brain capillary endothelial cells are connected together with continuous tight junctions. The results of transmissionelectron microscope show that: the structure and the permeability of BBB werechanged in each group, Aluminum induced the damage of cell membrane, organ, andthe tight junction of BBB. The permeability of BBB was enhanced when theconcentration of Aluminum was enhanced. Our immunochemistry and western blotresults showed firstly that: Aluminum induced the decrease of expression of F-actin andoccludin protein, this effect was related by the concentration of Aluminum, weconclude that the permeability and the ultrastructure of BBB changed by Aluminumwas contributed by the decreased expression of tight junction protein F-actin andoccludin.Zinc is an essential nutrient involved in many aspects of cell function. Forexample, Zinc participates in the metabolism of proteins, nucleic acids, carbohydrates,and lipids and is essential for cell proliferation and differentiation. Our study showedthat, appropriate dose of Zinc can protect the change of BBB induced by Aluminum, itcan accommodate the integrity of BBB which damaged by Aluminum. Our western blottest result showed that: mid-Al+low-Zn group vs. mid-Al group, high-Al+high-Zngroup vs. high-Al, the BBB tight junction protein expression were all significantly, butwere no significant difference when they compared with saline group. We can concludethat Zinc stabilized the cell membrane and increased the expression of occludin whichwere decreased by Aluminum.Although Zinc plays a considerable role on physiological function, highdoze-uptaken can induce negative effect. In our study, the BBB construction andpermeability were all changed in high-Zn group animals, there is a significantdifference between high-Zn group and saline group on the permeability of BBB.The decrease expression of tight junction protein occludin and BBB openingshowed that Aluminum toxicity may be related to the change of the permeability andthe integrity of BBB, this may be the original damage of CNS by Aluminum, whichmay induce the pathological change in a long stage. More attention should be paid on this. The structure of juvenile animals are not developed well yet, it can be influencedmore easily and be damaged badly. Further study is needed to get more informationabout Aluminum toxicity on BBB to gain new methods to protect and treat the metaltoxicity on juvenile animals.Conclusion1.10mg/kg Al influence the growth of juvenile rats, but not significant change onthe learning and memory ability.2. 2.5mg/kg Al exposure increases the permeability of the rats; the permeability,the ultrastructure, the expression of tight junction protein occludin and F-actin were allchanged when the dose of Al was enhanced.3.5mg/kg Zn has not influence the weight growth of the rats, and not changed thepermeability and the ultrastructure of BBB significantly; 10mg/kg Zn decreases theweight growth of the rats significantly, increases the permeability of BBB, also injuriesthe ultrastructure of BBB. This dose influenced the the expression of tight junctionprotein occludin and F-actin.4. Zinc can improves the brady-growth which induced by Aluminum, and protectsthe change of BBB induced by Aluminum inhibits the change of BBB tight junctionprotein occludin and F-actin expression significantly.