The current study constructed an amblyopia model by suturing the lid in the right eye of rat and performed hf-rTMS for 2 weeks. We found that the VIP expression in the visual cortex and the P100 amplitude significantly increased in amblyopic rats after intervention with hf-rTMS. The finding suggested that hf-rTMS could elevate the VIP expression in the visual cortex and improve the visual transduction function of rats with amblyopia.
Previous morphological and anatomic studies on amblyopia revealed that amblyopia was mainly attributed to a lesion in the visual cortex presenting with decline in synaptic plasticity and disorder of connectivity between neurons [11]. VEP provides an objective test of visual function [12] to reflect the connectivity between neurons. The occurrence of amblyopia is always accompanied by a significant decrease in VEP amplitude with or without extension of the latency period [13]. It has been proved that the absolute value of VEP amplitude is a frequently used criterion to quantify the severity of amblyopia and assess the therapeutic efficacy [14, 15]. The critical period for the development of binocular vision in rats usually is around 2–6 weeks of age [16]. In the present study, we selected rats at 3 weeks of age for MD by suturing the lid in the right eye. The P100 amplitude of the amblyopic eye was reduced while the latency was prolonged, suggesting successful modeling of amblyopia by form deprivation. Further analysis found that NC rats at 8 weeks of age had a significantly higher P100 amplitude than rats at 6 weeks of age, indicating age-related P100 amplitude, consistent with the previous study [17]. In addition, the P100 amplitude in MD rats also showed a similar changing trend. We speculated that single removal of deprivation has certain effects to improve amblyopia in rats, but further research is needed to confirm whether the rat age makes an effect.
rTMS is a neuromodulatory technique that can act on cerebral cortex to regulate neural excitability via generating a magnetic field. It is easy to operate, safe, and has few side effects, and thus it has been widely applied in clinical treatment of nervous system diseases, such as depression, seizure, and Parkinson’s disease (PD). It was first found as effective to temporarily improve the contrast sensitivity of the amblyopic eye of adult patients (returned to baseline after 1 week) by Thompson and Hess [5, 6], and it could be more effective in mild cases. To extend the effect, Clavagnie et al. [18] applied continuous theta burst stimulation (cTBS) for consecutive 5 days. They found that cTBS could long-term improve the contrast sensitivity of patients with amblyopia and stabilized it for up to 78 days, suggesting the long-term effect of cTBS on functional recovery from amblyopia. However, whether cTBS has promoting effect to other visual functions in patients with amblyopia was not explored in their study. Tuna et al. [19] examined the visual acuity, suppressive imbalance, and stereoacuity of amblyopic eye before and after cTBS and found significant improvement after cTBS. This result implied that TMS could change the visual cortical plasticity and concurrently strengthen the neural function of patients with amblyopia. Nevertheless, the majority of the current studies are limited to the subjective visual function associated with amblyopia without further exploring the potential molecular mechanism.
Previous research revealed that rTMS has certain effect to improve the function and structural plasticity of impaired synapse, such as increasing the synaptic curvature, the thickening of postsynaptic compact zone, the length of synaptic active zone [20, 21], the number of dendrites in neurons, the axon length, and the intercellular neural connections [22, 23]. The present study found that at 8 weeks of age, the P100 wave amplitude in the MD + hf-rTMS group was higher than that in the MD group but remained lower than that in the NC group. This result suggested that hf-rTMS affected the visual transduction function of rats with amblyopia via increasing neural excitability and improving synaptic connections between neurons.
It has been proven that amblyopia can lead to reductions of cerebral blood flow and metabolic function [24]. VIP was first found within the pig small intestine [25] and then proven with a wide distribution in cerebral cortex and lateral geniculate body [26]. It mainly acts as a neurotransmitter and neuromodulator [27] to help for vasodilation, increasing cerebral blood flow, and promoting cell proliferation and differentiation. In addition, it can also modulate the plasm level of hormones and affect the metabolic function of neurons [28]. Existing studies have also shown that activation of VIP-positive neurons can lead to gain of visual responses. For instance, amblyopia can induce paraptosis in visual cortical neurons [29], while VIP positive expression can inhibit the occurrence and development of amblyopia with antioxidant and anti-apoptotic activities [30, 31]. Consistently, our previous studies found that amblyopia resulted in down-regulated VIP expression in the visual cortex of kittens with amblyopia [8], and that exogenous introduction of VIP increased the VIP expression in the visual cortex and elevated the PVEP amplitude [32]. Combining the findings, VIP is critical to visual development.
hf-rTMS can elevate neuronal excitability and cerebral blood flow through inducing depolarization on the membrane potential of visual cortical neurons. Long-term hf-rTMS can trigger a series of physiological changes of neurons in turn to modulate synaptic plasticity, such as the release of excitatory and inhibitory neurotransmitters [33, 34] and increased expression of related proteins [35]. In rats, ~ 95% of the optic nerve axons cross in the optic chiasm to the contralateral side [36], which is different with the human. Therefore, the monocular amblyopia induced by form deprivation only involves the contralateral visual cortex. In the current study, the left visual cortex was selected for IHC and ISH analyses. We found that both the mRNA and protein expression of VIP in the visual cortex of rats were down-regulated after form deprivation, in agreement with the research mentioned above. Additionally, the mRNA and protein expression of VIP in the visual cortex were reversely increased following 2 weeks of hf-rTMS application, while it has been proven that elevation of VIP expression can physiologically improve visual functions. Research also reported that positive VIP expression can increase cerebral blood flow and nutritional support for neurons, and inhibit the paraptosis in neurons, thereby playing a neuroprotective role. Collectively, it could be inferred that hf-rTMS participates in the remodeling of the visual system via up-regulating VIP expression in the visual cortex.
One of the limitations of the present study is that this is a preliminary study on the mechanism of action of hf-rTMS with a single stimulation intensity, and its effect on neural transduction and VIP expression did not reach the levels found in healthy controls. We speculated that the full effects of hf-rTMS were not seen under such stimulation intensity. Further studies will be carried out to explore the effects of different stimulation intensities in rats with amblyopia.
To conclude, hf-rTMS can increase the VIP expression in the visual cortex and improve the neural transduction in rats with amblyopia, providing certain theoretical basis for amblyopia treatment with hf-rTMS. However, the specific pathways involved in visual cortical plasticity requires to be further clarified.
A participated in the design of the experiment, analyzed the data, and revised the manuscript. B participated in the establishment of animal models, specimen collection, experimental operations, data collection and analysis, and manuscript writing. C, D, and E participated in the creation of animal models, PVEP testing, specimen collection, and experimental operations. All authors read and approved the final manuscript.