At present, the classic method of modeling form deprivation amblyopia is upper eyelid suture [12,13,14]. In the past, our team selected 3-week-old kittens to establish amblyopia models by monocular form deprivation and used the Suture Covering Method [15], 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.
Some studies have shown that the dark environment will affect the plasticity of visual cortex in kittens, which will increase the amplitude of P100 waves and improve their eyesight [16, 17]. Therefore, in the process of establishing amblyopia kittens model, we only removed the black opaque covering cloth during detection. Moreover, we use natural light throughout the whole process to ensure the stability of their biological rhythm.
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 latency of P100 waves between different eyes at the same time and the same eye at different times, we found that P100 waves in three groups of PVEP detection, with the increase of age, the latency gradually shortened and the amplitude gradually increased. 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 schemes [18, 19]. The critical period of visual development in kittens is from 4 weeks to 16 weeks after birth [20, 21]. During this period, abnormal visual environment may lead to the influence of visual development. In the PVEP test, with the increase of age, the change of amplitude and latency of P100 wave in amblyopia eyes decreased compared with normal eyes. The changes of PVEP suggest that the appearance of form deprivation amblyopia in kittens makes various fine visual functions of visual system such as analytical perception, motion perception and stereo vision change. At present, some studies believe that establishing binocular stereo vision is one of the purposes of preventing and treating amblyopia [22]. The realization of this goal is based on synaptic plasticity.
Many neurotransmitters are considered to be related to the critical period of amblyopia and its plasticity changes [23, 24]. Moreover, in some studies, some proto-oncogenes are closely related to it [25, 26]. 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 activities. Among them, there is sufficient evidence to show the association between C-fos and amblyopia [13, 27]. The transcription factor of Egr-1 in the Egr family of immediate early genes is essential in the changes of visual cortex plasticity [28]. Egr-1 is a transcription factor encoding zinc finger structure, and its expression increases during synaptic plasticity, memory consolidation, LTP induction and learning [7, 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 the 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 system [28]. 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 functions [29, 30].
Immunohistochemistry and in situ hybridization were used to compare and analyze the expression of Egr-1 protein and mRNA in the visual cortex of 8-week-old amblyopia kittens and normal kittens. We found that positive cell numbers and the mean optical density of positive in the visual cortex of 8-week-old amblyopic kittens and normal kittens decreased. And the results show that there is a correlation among PVEP, IHC and ISH data. The appearance of this result provides strong proof for the association between Egr-1 and amblyopia. However, in terms of PVEP, although the amplitude of PVEP in the left eye of deprived kittens was lower than that in the right eye of the control group, there was no statistical difference between the two groups. Similar phenomena have been found in other studies [31]. Considering that the optic chiasma of cats is semi-chiasma, the change of visual cortex will affect both eyes. We speculate that this may be due to PVEP reflecting the conduction, transmission and excitation intensity of ocular ganglion cells to occipital visual cortex. Studies have shown that amblyopia is related to macular development and retinal fibrous layer thickness in addition to the changes of visual cortex-related protein expression [32,33,34].
The occurrence of amblyopia is related to synaptic plasticity changes, and long-term changes will occur in the process of amblyopia. The balance of excitation and depression at axonal level of the visual cortex is the condition of maintaining normal development and function of the visual cortex, and it is also an important factor affecting the plasticity of visual system [35]. The plasticity of nerve is mainly manifested in the plasticity of synaptic structure and function [36, 37]. 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 patients [5]. Perceptual learning can effectively restore the visual function through repeated visual stimulation and visual experience with supervision and feedback [38,39,40]. 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 animals [41, 42].
This study not only confirmed the correlation between Egr-1 protein and mRNA expression in visual cortex and amblyopia, that is to say, 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. But also indirectly verified the correlation between perceptual learning and amblyopia.
However, this study still has some limitations. In the process of modeling, we only use the random number table method to divide the kittens into two groups, but did not randomize the deprived eyes of the deprived kittens. On the other hand, we proved that the expression protein and mRNA of Egr-1 was down-regulated in the visual cortex of amblyopia kittens, but we failed to detect dynamically change of Egr-1, or set different groups to observe its changes with age.