| Avermectin (AVM) is a broad-spectrum antibiotic with anti-parasitic activity, it has been widely driven to kill agricultural pests and internal and external parasites of livestock in agricultural production and stock farming areas, subsequently, accumulation of AVM in the environment will also be increased. Excessive intake can cause severe nervous system damage in human and animals, the mechanism of AVM toxicity is growing concern by researchers. The pigeon is one of the preferred creature for monitoring changes in the ecological environment of biological, therefore, we chose the king pigeon as the object of study, establishing king pigeon sub-chronic toxicity model, observed the clinical symptoms of pigeon AVM poisoning, the histological structure of brain tissue, examined the contents of amino acid neurotransmitters in the pigeon brain tissue and the changes of DNA methylation level, besides, gene mRNA expression of amino acid neurotransmitter receptors, inflammatory cytokines, methyl transferases and demethylases, to explore the effect of AVM poisoning on king pigeon’s brain tissue and its toxicity mechanism, provide reliable data for further study of the effect of AVM on birds, and provide the scientific reference for the protection of the ecological environment and promote the development of animal husbandry.Ninety-six two-month-old healthy American king pigeons were randomly divided into four groups, twenty-four in each group, half male and half female, free feeding and drinking water, stepwise cage rearing, feeding in strict accordance with the feeding and management standard of king pigeon. King pigeons were fed with diets containing different doses of AVM (The control group was fed with a complete diet containing no AVM, AVM containing in the low dose group is20mg/(kg·diet), the middle dose group is40mg/(kg·diet), the high dose group is60mg/(kg·diet))to be exposed, copy king pigeon sub-chronic AVM toxicity model. The king pigeon were killed in the30th day,60th day,90th day, carefully separate the brain tissue of the king pigeon, take cerebrum, cerebellum and optic lobe tissue specimens for the detection of pathological observation, neurotransmitter and inflammation factors and other indicators. Application of high-performance liquid chromatography (HPLC) in detecting gamma-aminobutyric acid(GABA), Glycine(Gly), Glutamic acid(Glu), Aspartic acid(Asp) amino acid neurotransmitters and methylation levels in cerebellum, gene expression of amino acid neurotransmitter receptors gamma-aminobutyric acid A receptor(GABAAR), gamma-aminobutyric acid B receptor(GABABR), N-methyl-D-Aspartate, NMDA receptor subtypes(NR1, NR2A, NR2Breceptor) and inflammatory cytokines(cytokine inducible-NOS(iNOS), Nuclearfactor κB(NF-κB), prostaglandins E synthase (PGES), methyltransferases(DNMTs), DNMT1, DNMT3A, DNMT3B and demethylase (MBD2) in brain tissue was detected by real-time fluorescence quantitative PCR method, the results are as follows:1. Histological observation shows that the structure disorder of cerebrum, cerebellum and lobe tissue can be seen in AVM exposure groups compared with the control group, the main histological manifestations in cerebrum tissue is:around neurons appear space and small blood vessels, nerve fibers became disorder, neuron swelling change, cytoplasm increased, neuronal vacuolization and nuclear enrichment. The cerebellum and optic lobe tissue has the similar performance with cerebrum. Besides, cytoplasmic of Purkinje vacuolization, granular layer cells loose, concentrated or rupture, structural disorder and loose of granule cell nuclei, the gap increase between the nerve fiber can be seen in cerebellum. Ultrastructural observations show that compared with the control group, AVM exposure will lead to the damage to the ultrastructure of cerebrum, cerebellum and lobe tissue. The main ultrastructural changes in cerebrum tissue is incomplete nuclear membrane; condensed uneven chromatin distributed around in nuclear membrane or nuclear pore; mitochondrial swelling, mitochondrial ridge damage, dissolution and cavitation. The cerebellum and lobe tissue performe the similar with cerebrum. These confirm that AVM exposure can not only causes the clinical symptoms that the nervous system damage in King Pigeon, but also can lead to histological changes of the brain of King Pigeon, many parts of nerve cells’ultrastructure of King Pigeon’s brain was significantly damaged.2. Optimization of the high performance liquid chromatography detection method to detect the content of amino acid neurotransmitters and the level of DNA methylation of king pigeon brain tissues. Optimal conditions for detection of the content of amino acid neurotransmitters is: column:Diamonsil C18(4.6mm×200mm,5μm); the detection wavelength:350nm; mobile phase:A sodium acetate buffer,0.05mol/L(NaAc, pH=5.8), B acetonitrile-water(1:1, V/V), Gradient elution program B from13%of initial to48%after10min,18min to85%; velocity of flow:1.0ml/min; Column temperature is20℃; take10μl injection analysis. Optimization of chromatographic conditions to detect the level of DNA methylation are as follows:column: DIKMA SpursilC18-EP (250mm×4.6mm5μm); UV detection wavelength:280nm; mobile phase:methanol and0.05moL/L monopotassium phosphate(10:90, V/V, pH3.5); velocity of flow:1.0mL/min, the sample volume was20μ L. Using external standard calibration, UV detection. The follow-up results show that this method is rapid, accurate, simple, high sensitivity and good reproducibility.3. The content of amino acid neurotransmitters test results showed that the content of the four kinds of amino acid neurotransmitters Asp, Glu, Gly, GABA were all increased after AVM exposure(P<0.05), mRNA expression of GABAAR, GABABRNR1, NR2A, NR2B gene in the exposure group also significantly elevated compared with the control group(P<0.05), with exposure dose increase, levels of Asp, Glu, Gly, GABA showed a gradually increasing trend(P <0.05), GABAAR, GABABR, NR1, NR2A, NR2B gene expression levels also have the same variation trend. Variation trend in cerebrum, cerebellum and lobe tissue is similar, among which the most significantly increased content of amino acids neurotransmitter and receptor gene expression is in cerebrum tissue. This kind of change has a a certain dose-effect relationship with exposure time and dose of AVM. These show that AVM exposure caused increased content of four kinds of amino acid neurotransmitters in the King Pigeon’s brain tissue, the enhanced mRNA expression of its receptor genes may be one of the mechanisms of the neurotoxicity of AVM4. AVM exposure caused the total DNA methylation levels decreased in king pigeon brain tissue, the decrease of mRNA expression of methyl transferases DNMT1, DNMT3A, DNMT3B and the increase of mRNA gene expression of demethylase MBD2, and there is statistical significance compared with the control group(P<0.05), the cerebrum, cerebellum, optic lobe have the same variation tendency. With exposure dose increase, mRNA expression of DNMT1, DNMT3A, DNMT3B showed a gradually decreasing trend(P<0.05), mRNA expression of MBD2showed a gradually increasing trend(P<0.05). This kind of change also has a a certain effect relationship with exposure time and dose of AVM. These show that AVM exposure induced brain injury may be associated with decreased DNA methylation level.5. Effect of AVM exposure on the expression level of mRNA of iNOS, NF-κB, PGES gene in the cerebrum, cerebellum, optic lobe is similar. In the30th day, the mRNA expression of these genes gradually increased in a dose-dependent manner(P<0.05), but in the60th day and the90th day, the mRNA expression of these genes shows the first rise and then decline(P<0.05). In the cerebellum and optic lobe the expression of these inflammatory cytokines showed a similar trend. The changes levels perform different in different brain areas and time point. These suggesting that inflammatory genes iNOS, NF-κB, PGE participated in the brain tissue’s toxicity damage process induced by AVM.In summary, the damage change can be seen both in the cerebrum, cerebellum and lobe tissue after AVM exposure, from which the most notably change may be the cerebrum tissue, this suggests that the cerebrum may be one of the main parts of AVM poisoning causing damage. This test is based on light and electron microscopic observation of pathologic morphology, by detecting the content of the amino acid neurotransmitters in brain tissue and the expression of related receptor gene, gene expression of inflammatory cytokines, the DNA methylation level and the gene expression of the methyl transferases and demethylase, to explore and enrich the neurotoxicity mechanism of AVM. |
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