ABSTRACT
Producing polyclonal antibodies (IgY) in chickens has advantages over those obtained in other animal models, since they have been used as a tool for studying different proteins (NMDA glutamate receptor in our case, specifically the NR1 subunit). We produced specific antibodies against expression products by the alternative splicing of the gene encoding NMDA receptor NR1 subunit in adult rat brain. Three peptides corresponding to the splicing sites (N1, C1 and C2' cassettes) were designed, synthesised and used individually as antigens in hens. Specific immunoglobulins were purified from yolks. The antibodies were then used for purifying the NMDA receptor NR1 subunit using affinity chromatography coupling the three antibodies to the support.
La producción de anticuerpos policlonales en gallinas (IgY) tiene ventajas sobre anticuerpos obtenidos en otros modelos animales y se han empleado como una nueva herramienta para estudiar diferentes proteínas (el receptor de glutamate tipo NMDA en nuestro caso, específicamente la subunidad NR1). Produjimos anticuerpos específicos contra productos de expresión por splicing alternativo del gen que codifica la subunidad NR1 del receptor tipo NMDA en el cerebro de rata adulta. Se diseñaron 3 péptidos correspondientes a los sitios de splicing del gen (conocidos como casetes N1, C1 y C2'), se sintetizaron y se usaron individualmente como antígenos en gallinas. Inmunoglobulinas específicas se purificaron de las yemas. Los anticuerpos se usaron para purificar la subunidad NR1 del receptor tipo NMDA usando cromatografía de afinidad, a través del acople de los tres anticuerpos al soporte.
A produção de anticorpos policlonais (IgY) em galinhas tem vantagens sobre os obtidos em outros modelos animais e eles têm sido usados como uma ferramenta para o estudo de proteínas diferentes (NMDA receptor de glutamato no nosso caso, especificamente a subunidade NR1). Nós produzimos anticorpos específicos contra produtos de expressão pela splicing alternativo do gene que codifica receptor NMDA subunidade NR1 no cérebro de ratos adultos. Três peptídeos correspondentes aos locais de splicing (N1, C1 e C2' cassetes) foram concebidos, sintetizados e utilizados individualmente como antígenos em galinhas. Imunoglobulinas específicas foram purificadas a partir de gemas. Os anticorpos foram então usados para purificar o receptor NMDA subunidade NR1 utilizando cromatografia de afinidade, por meio da junção dos três anticorpos ao suporte.
ABSTRACT
Objective To evaluate the effects of electromagnetic irradiation of 2000 μW/cm2 exposure on mRNA and protein expression levels of immunoreactive protein and mRNA of NMDAR1 in rats hippocampal,and to explore the impaired mechanism of electromagnetic irradiation on learning and memory.Methods Rats were randomly divided into normal control group, sham-radiated group, and 1 h/d, 2 h/d, and 3 h/d radiation groups.The rats in the radiation groups were fixed and recieved microwave exposure of 2000 μW/cm2, then their learning and memory abilities were tested by Morris water maze experiment, the change of NR1 protein in hippocampal neurons of each group of rats was measured with immunohistochmistry and western blot techniques, and the expression of NR1 mRNA in hippocampus was determined by RT-PCR.Results In the water maze test,compared with the normal control group (8.8 ± 1.66 ), the escape latency of three radiated groups rats ( 1 h/d ( 12.29 ±1.36) s,2 h/d ( 17.99 ±2.25) s,and 3 h/d (24.66 ±5.56) s) were significantly longer (P＜0.05).In the radiation group,the hippocampal neurons of rats showed evident reduction in the ratio of NR1 positive cells,irregular,and arrayed in disorder.Moreover,compared with the normal control group ( (0.70 ±0.11 ), (0.68 ±0.11 ) ) ,the expession of NR1 protein ( 1 h/d (0.122 ±0.026) ,2 h/d (0.102 ±0.023) ,and 3 h/d (0.060 ± 0.009) ) and its mRNA ( 1 h/d (0.46 ±0.07) ,2 h/d (0.35 ±0.05) ,and 3 h/d (0.12 ±0.02) ) in hippocampal neurons was significantly decreased (P＜0.05).Among the indicators, there was no significant difference between sham-radiated group and normal control group.Conclusions Electromagnetic irradiation of 2000 μW/cm2 exposure can impair the learning and memory abilities of rats possibly through a mechanism correlated with the lower expression of NR1 protein and its mRNA in hippocampus.
ABSTRACT
Objective To observe the expression and phosphorylation of N-methyl-D-aspartate (NMDA) receptor NR1 subunit in rat cortex and hippocampus under high altitude hypoxia. Methods The adult SD rats were exposed to hypobaric hypoxia imitating 5 500-meter high altitude for 8 h daily for 3, 7, 14, 21 d. Immunohistochemical staining, Western blotting and co-immunoprecipitation were used to detect the expression and phosphorylation of NR1 in rat cortex and hippocampus. Results Both immunohistochemistry and Western blotting showed NR1 expression in rat cortex and hippocampus was increased under hypoxia in a time-dependent manner. The tyrosine phosphorylation of hypoxia groups was increased, and reached the peak on day 14 after hypoxia, then decreased, still higher than that of control groups till day 21 (P
ABSTRACT
Objective To investigate the mechanisms of the inhibitory effects of peripheral electrical stimu- lation(PES)on chronic central pain(CCP)after spinal cord injury(SCI).Methods Twenty-four male Sprague- Dawley rats with CCP following SCI were randomly divided into three groups:a group without stainless steel needles implanted (NSSN group,n=8),a group with a stainless steel needle implanted but no peripheral electrical stimula- tion applied(NPES group,n=8)and a PES group(PES group,n=8).The rats' CCP was evaluated through ob- serving their response to nociceptive stimulation by means of the paw withdrawal pressure threshold(PWPT)and the paw withdrawal latency(PWL).Spontaneous pain behaviors including autophagia and scratching were observed at the same time.PES was applied via stainless steel needles inserted into standard acupoints on the hind limps and the back.The expression of the NMDA receptor 1(NR-1)subunit in the spinal cord horn was measured using immuno- chemical methods.Results Compared with the NSSN and NPES groups,CCP in the PES group was alleviated, PWPT and PWL were dramatically increased(P＜0.01)and the expression of NR-1 was obviously decreased (P＜0.01).Conclusion Peripheral electrical stimulation may alleviate chronic central pain after spinal cord injury in rats.