Form deprivation amblyopia is caused by congenital cataract, ptosis, corneal leukoplakia and other reasons in infancy, which makes light unable or abnormal to enter the eye and deprives the macula of the opportunity to receive normal light stimulation12–14. The reduction of physiological stimulation causes macular dysplasia or stagnation during development13. For kittens, the effect of form deprivation on their visual acuity reached its peak from postnatal to 4 weeks old. After 12 weeks old, form deprivation caused by covering could hardly interfere with their visual acuity development15.Therefore, we selected 3-week-old kittens to establish amblyopia models by monocular form deprivation. At present, the classic method of modeling form deprivation amblyopia is upper eyelid suture16–18.
In the past, our team used the the Suture Covering Method19, that is, using black covering cloth instead of eyelid suture to model amblyopia. The continuous observation of PVEP changes in experimental animals was realized by suture while preventing eye injury caused by eyelid suture. This method can effectively block the light from covering the eyes of animals in all directions and successfully establish amblyopia animal models. However, during the modeling period, some kittens still had head movements, which led to light passing through the side of the covering cloth. In order to improve this situation, this experiment increased the diameter of the covering cloth in the original covering method, and made a black opaque covering ring with a certain height in the center of the covering cloth to further enhance the covering efficiency.
In this experiment, checkerboard flip stimulation was used to detect PVEP, and the wave images of PVEP in kittens were recorded, which were composed of two positive waves and one negative wave, showing an "M" shape. By comparing the amplitude and incubation period of P100 wave between different eyes at the same time and the same eye at different times, we found that the amplitude of P100 wave decreased and the latency of P100 wave increased in the right eye of deprivation group compared with the left eye of deprivation group and the right eye of control group. This is similar to the previous amblyopia modeling results by other schemes20, 21. The results of this study suggest that the improved covering method is feasible and effective. The animal model of monocular form deprivation amblyopia can be successfully established by the the Suture Covering Method, and the dynamic detection of eye physiological parameters of experimental animals can be realized.
With the development of amblyopia research in recent years, many neurotransmitters involved in the plasticity mechanism of visual development, such as AMPA receptor and its GluR2 subunit, NEP1-40, synaptophysin, cholinergic neurons, growth-associated protein-43, GABA, cPKC-r, NMDAR, NGR-1, etc22–27, are considered to be related to amblyopia, but the relationship between the critical period of amblyopia and its plasticity changes cannot be completely explained at the molecular level27, 28. As a class of proto-oncogenes encoding transcription factors, immediate early genes have the effect of coupling short-term signals with long-term changes, mainly including C-fos, Egr family and Arc, among which many transcription factors are regulated by visual activities29, 30. Among them, there is sufficient evidence to show the association between C-fos and amblyopia17, 31. The transcription factor of Egr-1 in the Egr family of immediate early genes is essential in the changes of visual cortex plasticity32. Egr-1 is a transcription factor encoding zinc finger structure, and its expression increases during synaptic plasticity, memory consolidation, LTP induction and learning7, 8. Cytoskeleton related gene Arc, as one of the target genes of Egr-1, is an effector molecule induced by synaptic activity and plays an important role in late LTP. Studies have shown that under certain conditions, Egr-1 can regulate the transcription of late activity-dependent Arc gene in hippocampus CA1 region, and the immediate early gene Arc can connect the change pattern of neural activity and synaptic plasticity because of its pluripotent and fine tuning system, thus optimizing the information storage of nervous system32. In addition, previous studies on amblyopic animal models have found that there are structural changes in ganglion cells, lateral geniculate body and visual cortex of amblyopic animals, accompanied by a decrease in synaptic density, which will lead to further changes in their functions2, 33, 34.
Immunohistochemistry and in situ hybridization were used to compare and analyze the expression of Egr-1 protein in visual cortex of 8-week-old amblyopia kittens and normal kittens. The results of this experiment suggest that during the critical period of visual development, due to the unequal input of binocular vision, the number and morphology of visual cortex cells may change, resulting in the decrease of Egr-1 protein and mRNA expression, which further affects the normal physiological function of visual cortex and affects visual development, thus promoting the occurrence and development of amblyopia.
The balance of excitation and depression at axonal level of visual cortex is the condition of maintaining normal development and function of visual cortex, and it is also an important factor affecting the plasticity of visual system35. The plasticity of nerve is mainly manifested in the plasticity of synaptic structure and function36, 37. The occurrence of amblyopia is related to synaptic plasticity changes, and long-term changes will occur in the process of amblyopia. Egr-1 and Arc genes are closely related to long-term changes of synapses. At present, most studies also show that perceptual learning can improve the visual function of amblyopia patients5. Perceptual learning can effectively restore the visual function through repeated visual stimulation and visual experience with supervision and feedback38–40. Researchers found that its mechanism may be related to the balance of excitement and depression related to plasticity, and Egr-1 and Arc play an important role in learning and memory of humans and animals41, 42. This study not only confirmed the correlation between Egr-1 protein and mRNA expression in visual cortex and amblyopia, but also indirectly verified the correlation between perceptual learning and amblyopia.