| BackgroundAmblyopia is caused best corrected visual acuity decline(≤0.8)in monocular or binocular by improper visual experience at the critical period of visual development, which examined without organic diseases in eye. It is conveyed that amblyopia affects approximately 1.6%～3.6% of the general population and the pathogenesis is not fully clear, the treatment focuses on monocular occlusion in clinical. In recent years, the role of synaptic plasticity in visual neuronal at the critical period of visual development has become the forcus. But the related research has been made in comprehending the molecular components at the postsynaptic membrane such PSD-95, and lacking the research on morphology and function in the presynaptic ending. Synaptophysin (SYP) is the major integral membrane protrin accounting for 6%～10% of total synaptic vesicle protein, playing an important role in many physiological activities such as axonal growth and synaptogenesis, relaease neurotransmitter, memory and learning, synaptic plasticity and so on. Studies have inferred that the SYP may take part in early development and maturation in visual cortex, enriched environment (EE) can re-activate the visual cortex again of adult amblyopia rats. However, the expression of SYP during the critical period in visual cortex, and what influence of reverse suture (RS) and EE have effected on the expression of SYP have not reported so far.ObjectiveIn this subject, we attempt to explore the expression of SYP in the visual cortex of monocular deprivation amblyopia rats before and after reverse suture and enriched environment treatment in the critical period, and to explore the influences of SYP has effected on the visual neuronal synaptic plasticity.Methods1 Seventy-two SD rats aged 14 days old, male and female unlimited(clean animals, offered by animal experimental center of Xinxiang Medical College), were randomly separated into 6 groups and 12 rats in each group:The normal control groups include normal control group Ⅰ(NCI) and normal control group Ⅱ (NCⅡ), the monocular deprivation groups include monocular deprivation group Ⅰ(MDI)、monocular deprivation group Ⅱ(MDⅡ)、reverse suture group(RS) and reverse suture plus enriched environment group(RS+EE).For the 4 groups of MDⅠ、MDⅡ、RS、RS+EE sutured right eyelid respectively in each group, MDI group at 28 days of age、MDⅡ group at 42 days of age, tested by pattern visual evoked potential (P-VEP); RS and RS+EE group at 28 days of age to test P-VEP while open the deprived eyes and at the same time to suture the other eyelid for the treatment of covering, Rats of RS group were raised understand the standard environment and rats of RS+EE group were housed in the EE, after 14 days (42 days of age) tested by P-VEP again for the deprived eyes. The 2 groups of NCI and NCII did not make any processing, NCI group up to 28 days、NCⅡ group up to 42 days put to test P-VEP. Each of group put to priming and material after testing by P-VEP.2 Using HE staining to observe the changes of neuron morphology in the visual cortex of each group rats, applying immunohistochemistry staining and Real Time PCR skill to measure the expression of SYP and the SYP mRNA transcription in the visual cortex of different group rats.3 Observe the expression of SYP in visual cortex by the optical microscope, image capture at the same light intensity and amplification, observe the immune average optical density(AOD) of SYP in visual cortex by using the Auto-Slide ICM. Analysis the record by SPSS 19.0 statistics software, compare difference between groups at 28 days of age(NCI and MDI group) by paired-sample t test; difference between the 4 groups at 42 days of age(NCⅡ、MDⅡ、RS and RS+EE group) using single-factor analysis of variance and analysis of LSD test. The differences have significantly statistics (P<0.05).Results1 The P-VEP results:The P-VEP of NCI and NCⅡ group have steady wave-forms, latency and amplitude; Compared with the deprived group rats with the same age(MDI, MDⅡ), the degree of latency extended and amplitude of the Pioo wave were decreased(P<0.01); There are no significant differences of wave-forms、latency and amplitude between normal eyes of NC group rats and undeprived eyes of MD groups with the same age(P>0.05); Contrasted to MDⅡ with the same age, the degree of change for instability, latency decreased and amplitude of the Pioo wave in RS group were rised(P<0.05); while to NCⅡ group there still have differences(P<0.05); Contrasted to MDⅡ and RS group with the same age, the degree of change was unstable, latency decreased and amplitude of the Pioo wave in RS+EE group were rised(P<0.05); while contrasted to NCⅡ group, there has no significantly different (P>0.05).2 The HE staining results:The visual cortex neurons of rats in NCI and NCⅡ group were layered distribution, cell shape, nuclear is dyed blue, cytoplasmic staining is red, the neurons arrangement were order of each layer; Compared with NCI and NCⅡ group, there were no apparently abnormality of MDⅠ、MDⅡ、RS and RS+EE group in visual cortex.3 The outcome of immunohistochemistry:The immunopositive products of SYP in visual cortex were granular and dotted with tan, and specificially expressed in terminal of the synaptic in neuron cells. The SYP positive products expressed in all layers of area 17 in visual cortex, while there had no specific staining in all of NC groups. The AOD of SYP positive products in the visual cortex of MDI group was obviously less than those in NCⅠ group with the same age (t=4.074, P<0.01); The AOD of SYP positive products in the visual cortex of MDⅡ group was obviously less than those in NCⅡ group with the same age (t=7.544, P<0.01);The AOD of SYP positive products in visual cortex have significantly difference among NCⅡ、MDⅡ、RS and RS+EE group(F=25.503, P<0.001) with the same age; the RS+EE group was higher than those of RS group(P=0.029), the RS group was higher than those of MDII group(P<0.001), the RS and MDII group were lower than NCII group(P<0.05),but the difference of RS+EE group contrasted to NCII group at the same age has no obviously different (P=0.756).4 The results of RT-PCR:The expression of SYP in the visual cortex incd all group rats. The comparative expression of SYP mRNA in visual cortex of MDI group was obviously much less than those in NCI group with the same age (t=5.608, P<0.05); The comparative expression of SYP mRNA in visual cortex of MDII group was obviously much less than those in NCII group with the same age (t=7.559, P<0.01); The expression of SYP mRNA in visual cortex have significantly difference among NCⅡ、MDⅡ、RS and RS+EE group(F=33.556, P<0.001) with the same age; the RS+EE group was higher than those of RS group(P=0.003), the RS group was higher than those of MDII group(P<0.001), The expression of SYP mRNA in visual cortex of MDII and RS group were lower than NCII group with the same age (P<0.05); but there has no significant difference between RS+EE and NCII group with the same age(P=0.693).Conclusion1 In visual development of the critical period, Monocular deprivation reverse suture and enriched environment treatment might influence the expression of SYP in the visual cortex, and imply that SYP protein might affect the plasticity of neurous and participate in the the progress of plasticity in visual development. SYP protein was one of the key moleculars in the mechanism of amblyopia pathogenesis.2 The cover treatment and enriched environment may partially reverse the neural activity of deprived amblyopia in the visual cortex during the critical period. It also offered the theoretical foundation for curing children with amblyopia in the clinic. |
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