The Effect Of PAS-Na To GABAaegic Neurotransmitter In Basal Ganglia Of Mn-exposed Rats

Manganese (Mn) is a naturally occurring element that is an essential nutrient, and cofactor for certain metalloenzymes required for normal cellular homeostasis. Although Mn consumption is necessary in humans, excessive exposure is associated with body's adverse effects, in especial the psychiatric, cognitive, and motor adverse effects. Magnetic resonance imaging studies are in agreement that Mn accumulates to a large extent in the globus pallidus. The globus pallidus and related basal ganglia structures have therefore been a focus of the preponderance of the research efforts on Mn neurotoxicity. it is now believed that the GABAergic system is probably perturbed first in the etiology of manganism. Several reports have linked Mn exposure to decrease or increase in the concentration of brain GABA.Addressing a need for a mechanism to explain the apparent reduction in GABA concentration. Reported decreases in overall brain Glu decarboxylase (GAD) and GABA-transaminase (GABA-T) levels subsequent to intraperitoneal injections of MnCl2, and alterations in expression of GABA transport and receptor proteins.Sodium para-aminosalicylic acid (PAS-Na) is effective in reducing Mn concentrations in body fluids and brain to physiological level, thus acting as a Mn-chelating agent. In clinical studies and obtained animal studies have found that the PAS-Na can get rid of brain manganese, repair manganese-induced brain damage and ultrastructural changes, the body's enzymes returned to normal, the antagonistic manganese-induced learning and memory changes, oxidative stress change, neural microfilament protein(NF) positive cells of the dentate gyrus granulosa cells and the glial fiber acid protein (GFAP) positive in the hippocampus.Meanwhile the PAS-Na used in the treatment of clinical manganism patients were also found in patients with a variety of symptoms and signs such asneurasthenic symptoms, eyes closed, elite refers to nose test, refer to the test write barriers have improved markedly, and the efficacy of the consolidation of follow-up after17years of clinical effect is still good. However,The mechanism of PAS-Na for manganese exposure caused the GABAergic damage still unknowed. This study intends to explore the intervention mechanism of PAS-Na exposed to manganese for the basal ganglia GABAergic by short-term and subchronic Mn exposed rats in vivo.[Objective] To study the role of sodium aminosalicylate (PAS-Na) on growthing development, learning and memory, the ultrastructure of the basal ganglia and the concentration of GAB A, activity of GAD and GABA-T, GABA A receptor (GABAAR) and GABA transporter (GAT) expression in basal ganglia of short-term and subchronic Mn exposed rats.[Methods and materials](1) Animals grouping and exposure. Sprague-Dawley male rats received daily intraperitoneally (i.p.) injections of15mgMn/kg,5days/week for4weeks, followed by a daily subcutaneously (s.c.) dose of PAS-Na (100and200mg/kg as the PAS-L and PAS-H group, respectively) for another3or6weeks. Sprague-Dawley rats received daily i.p. injections of15mg Mn/kg,5days/week for4,8orl2weeks, followingby a daily s.c. dose of PAS-Na200mg/kg as the PAS-P group,3days/week at the same time for8and12weeks, followed by a daily s.c. dose of PAS-Na (100,200and300mg/kg as the PAS-L, PAS-M and PAS-H group, respectively) for another6weeks. the rest of the groups sc an equal volume of saline.(2) determination of indicators①Growthing development indicators detection. Weighing the anminal weight one time every week. Obtaining the organ of heart, liver,spleen,lungs,kidneys,testis, and weighing the weight of them,than calculate its organ body ratio.②The learning and memory ability test. Before the anatomy, Morris watermaze test lasting for6days was used to detect learning and memory ability of rats.③The Ultrastructural of the basal ganglia observation. The end of theexperiment, five animals of each group perfused the mixed liquor of2%paraformaldehyde and2.5%glutaraldehyde through the heart, than separated the basal ganglia, observed ultrastructure changs by transmission electron microscopy④GABAergic neurotransmitters detection. At the end of the experiment,the animals were sacrificed to take the basal ganglia rapidly and froze in liquid nitrogen and stored at-80℃. To survey the content of GAB A, Glu, Gln and Gly by high performance liquid chromatography (HPLC) with fluorescence detection.To examine the activity of GAD and GABA-T by high performance liquid chromatography and fluorescence spec trophotometer detection.To probe the mRNA and protein expression of GABA transporters and GABA receptors (GABAAR) by real-time fluorescent quantitative PCR (RT-PCR) and Western blot (WB).[Results]1. Effects of PAS-Na to growthing development of Mn exposure rats.①Weight gain changes. The weight of the rats in each group with time growthing in all of our experimental period. Manganese exposed rats growth slower than the control rats. From exposed to manganese to the fourth week, eighth and tenth week, the manganese exposed rats body weight growed signafiance slower than the control rats. The weight of preven rats growed signafiance faster than Mn exposure rats from the first to the third week. There are no no significant change in rats bod's weight by the remedy of PAS-Na after Mn exposure, have nothing to do with the length of the period of PAS-Na treatment and Mn exposure.②The ratio of organ to body. Regardless of the length of period exposed to manganese, the rat showed the increasing of spleen quality. The rat exposed to manganese for4weeks, not only caused the increasesing of spleen quality but also the testis. When menganese exposure for12weeks, in addition to the spleen quality significantly increased, the quality of liver also significantly increased. Prevention with PAS-Na for12weeks to12weeks manganese-exposed rats, may recove the quality of liver and spleen. Treatment with L-PAS and H-PAS for6weeks to12weeks manganese-exposed may restore the liver body ratio.2. Effects of PAS-Na to learning and memory abilities of Mn exposure rats.①The effect on learning capability.The rats whether exposed to Mn for4weeks,8weeks,12weeks or were Mn-exposed for4weeks then stop3weeks,6weeks and were Mn-exposed for12weeks then stop6weeks, their incubation period and swimming journey increased compare with control. The effects of PAS-Na on learning and memory capabilities in Mn-exposed rats Whether in exposed to Mn for4weeks then treated with PAS-Na for3weeks or6weeks,or were Mn-exposed and PAS-Na prevented at the same time for8weeks or12weeks,or exposed to Mn for12weeks then treated with PAS-Na for6weeks the incubation period and swimming journey were decreased.②The effect on memory capability. The platform quadrant time or distance to total time or total distance ratio. With4weeks of Mn-expose then PAS treated for3weeks increased compare with control; contrary, in H-PAS group decreased compare with Mn-exposed rats. Mn-exposed and PAS-Na prevented at the same time for8weeks, the platform quadrant time or distance to total time or total distance ratio in Mn-exposed group rats decreased compare with control,and PAS-prevented group increased compare with Mn-exposed group rats.3. Effects of PAS-Na on basal ganglion ultrastructure of Mn-exposed rats.Short-term and subchronic Mn-exposed may induced basal ganglion damaged, neuropil, astroglia cells damaged. caused the neurons appeared degeneration, apoptosis and necrosis, that neurons dendritic and axon terminal swelling, astroglia cells appeared hyperplasia and apoptosis degeneration, and with the extension of Mn-expose, the damage become more serious. PAS-Na could against the alteration of basal ganglion in Mn-exposed rats, restored the degeneration, apoptosis and necrosis in neurons, synaptic cleft clear, but there was no significant in the basal ganglion of rats were exposed to Mn for12weeks.4. Effects of PAS-Na on GABAergic neurotransmitter in basal ganglion of Mn-exposed rats(1)The effects of Short-term and subchronic Mn-exposed on GABAergic neurotransmitters in basal ganglion. Mn-exposed for4weeks, the concentrations of Glu,Gln and GABA, the GAD activity and GABAAR mRNA expression significant increased in basal ganglion. Mn-exposed for4weeks and then stop for3weeks, the concentrations of Gly decreased and GABAAR protein expression increased significantly. When stop exposure to Mn for6weeks, the concentrations of Glu,Gln,Gly and GABAAR and GAT-1mRNA expression significant decreased. Mn-exposed for8weeks, the ratio of Glu/GABA and GABAAR mRNA expression decreased significantly. When stop exposure to Mn for12weeks, GABA-T activity and GABAAR protein expression increased, and stop Mn-exposed for6weeks, the concentrations of Glu,Gln,GABAand the expression of GABAAR mRNA decreased, and GAD and GABA-T activities, GAT-1mRNA expression increased.(2) The prevention and/or treatment effects on Short-term and subchronic Mn-exposed rats in basal ganglion. ①The effect of treatment with PAS-Na. After Mn-exposed for4weeks then used L-PAS and H-PAS treated for3weeks, the concentrations of Gly and GABAAR protein expression alterations no recovery. When for6weeks treatment, the changes of the concentrations of Glu, Glu and GAT-1mRNA protein expression were restored by L-PAS and H-PAS, H-PAS may returned the Gly concentration, but no effects on GABAAR mRNA expression no matter by L-PAS or H-PAS. When Mn-exposed for12weeks rats than treatment withL-PAS,M-PAS and H-PAS for6weeks, the alteration of GAD activity recovered by M-PAS and H-PAS, the alterations of GABA-T activity and GAT-1mRNA expression both restored by H-PAS,but Glu, Gln and GABA content and GABAAR protein expression did not recovered by any PAS-Na dose treatment.②The effects of prevention with PAS-Na. Mn-exposed and PAS-Na prevented at the same time for8weeks, the changes of Glu/GABA ratio and GABAAR mRNA expression recovered. Mn-exposed and PAS-Na prevented at the same time for12weeks, the alter of GABAAR protein expression restored, but no the GABA-T activity.[Conclusion](1) Short-term and subchronic Mn-exposed could lead to the slower growth of weight vs the control. Results showed that Mn has the growthing development toxicity. Prevention with PAS-Na in early period of manganese exposure, could restore the weight changing caused by manganese exposure.(2) Exposure manganese in short-term could affect the rat testes quality and the quality of the spleen, and in long-term exposure not only affect the spleen quality, but also the liver quality,l ead to its organ body ratio rising. The prevention of PAS-Na in organ quality changed caused by manganese exposure is good.(3) Short-term and subchronic manganese exposure all could affect the rat learning ability, and the memory fuction change only appeares in the long term period of manganese exposure. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in learning and memory fuction.(4) Short-term and subchronic manganese exposure all could affect basal nuclear ultrastructure. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in basal nuclear ultrastructure.And the treatment effect is better in the early period manganese exposure,the prevention effect better than the treatment.(5) Short-term and subchronic manganese exposure could affect the content of Glu, Gln, Gly and GAB A, GAD and GABA-T activity, GABAAR mRNA and protein expression and GAT-1mRNA in basal nuclear. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in that alteration. PAS-Na plays a prevention effect in GABAAR mRNA or protein expression of subchronic Mn-exposed rats, and a treatment effect in the content of Glu, Gln and Gly, GAD and GABA-T activity and GAT-1mRNA expression of Mn-exposed rats.