ARC/Arg3.1 expression in the lateral geniculate body of monocular form deprivation amblyopic kittens

doi:10.21203/rs.3.rs-2089824/v1

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Research Article

ARC/Arg3.1 expression in the lateral geniculate body of monocular form deprivation amblyopic kittens

https://doi.org/10.21203/rs.3.rs-2089824/v1

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Journal Publication

published 03 Jan, 2023

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Purpose

The present study compared the expression of activity-regulated cytoskeleton-associated (ARC/Arg3.1) in the lateral geniculate body between form deprivation amblyopia kittens and normal kittens to examine the significance of ARC/Arg3.1 in the lateral geniculate body in the pathogenesis of amblyopia.

Methods

Twenty kittens were randomly divided into experimental group (n = 10) and control group (n = 10). Black opaque covering cloth was used to cover the right eye of kittens in the experimental group. Pattern visual evoked potentials (PVEP) were detected weekly in all kittens. The expression of ARC/Arg3.1 gene was detected by immunohistochemistry and in situ hybridization, and apoptosis of lateral geniculate body cells was detected by TUNEL.

Results

PVEP detection showed that at the age of 5 and 7 weeks, the latency of P100 in the right eye of the experimental group was higher than that of the other three groups (P < 0.05), and the amplitude of P100 was lower than that of the other three groups (P < 0.05). The expression of ARC/Arg3.1 protein (P < 0.05) and mRNA (P < 0.05) in the lateral geniculate body of the experimental group was significantly lower than that of the control group. And the level of neuronal apoptosis in the experimental group was higher than that in the control group (P < 0.05). The expression of ARC/Arg3.1 gene was negatively correlated with the apoptosis level of lateral geniculate body neurons.

Conclusions

The expression of ARC/Arg3.1 is associated with monocular form deprivation amblyopia and apoptosis of lateral geniculate body cells.

Amblyopia

Activity-regulated cytoskeleton-associated (ARC/Arg3.1)

Lateral geniculate body

Form deprivation

With the in-depth exploration of the pathogenesis of amblyopia in molecular biology, neurobiology and other disciplines, related studies believe that visual developmental plasticity is the basis of amblyopia treatment, and its performance is closely related to synaptic plasticity^{1, 2}.

Synapse, as the structural basis of information transmission between neurons, is the key part of visual development plasticity³. In many pathogenesis of amblyopia, synaptic plasticity is considered as the most critical link and may be the final pathway of other pathogenesis. And its plasticity can be divided into long-term potentiation (LTP) and long-term depression (LTD) according to time^{4, 5}. The immediate early gene, activity-regulated cytoskeleton-associated (ARC/Arg3.1) is induced in neurons in response to neural activity and is necessary for activity-induced forms of synaptic plasticity⁶. Moreover, it also is a crucial regulator of memory and cognitive flexibility⁷. Previous studies have shown that there are structural changes in ganglion cells, lateral geniculate body and visual cortex of amblyopia animals, accompanied by a decrease in synaptic density. In the lateral geniculate body and visual cortex, apoptosis in amblyopic animals was higher than that in normal animals^8–10.

So, is there a certain connection between ARC/Arg3.1 and amblyopia? However, there is no article on the relationship between them yet. Therefore, by detecting the difference in the expression of ARC/Arg3.1 in the lateral geniculate body between amblyopic animals and normal animals, we explored the significance of lateral geniculate body ARC/Arg3.1 in the pathogenesis of amblyopia, and provided theoretical support for the occurrence and development of amblyopia.

Animals

A total of 20 healthy domestic cats were selected. All kittens were examined to rule out congenital and developmental abnormalities such as refractive media and fundus opacity. At the same time, there was no statistical difference between the two groups of kittens in terms of baseline diopter, axial length, pattern visual evoked potential (PVEP). (Table 1)

Table 1

Comparison of baseline situation between two groups of cats. (± S)
Group	right eye of the experimental group	left eye of the experimental group	right eye of the control group	left eye of the control group	F	P
Axial length (mm)	12.08 ± 0.32	12.07 ± 0.23	12.05 ± 0.202	12.19 ± 0.32	0.509	0.679
Diopter (D)	2.93 ± 0.49	3.00 ± 0.41	2.83 ± 0.50	2.78 ± 0.43	0.483	0.696
P100 latency (ms)	118.66 ± 3.10	118.36 ± 4.11	117.04 ± 2.41	117.27 ± 2.85	0.629	0.601
P100 amplitude (µV)	4.995 ± 0.661	5.073 ± 0.854	5.027 ± 1.033	5.051 ± 1.081	0.013	0.998

All kittens were kept in a room with an ambient temperature set to 24 ± 1°C and a relative humidity of 50 ± 10%, which had great ventilation and natural light. Before the age of 5 weeks, the kittens were given milk and water 5 times a day because the kittens could not eat independently. After one week of age, give kittens plenty of food and water three times a day. This study has been approved and supervised by the Experimental Animal Ethics Committee of North Sichuan Medical College (NSMC Appl. No. 2021 [66]), and all animals in this study were provided by the Experimental Animal Center of North Sichuan Medical College.

Animals model establishment

Twenty kittens were used the random number table method to divide into the experimental group (n = 10) and the control group (n = 10). The kittens were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). The right eye of the kittens in the experimental group was covered with black opaque covering cloth, while the control group was only anesthetized. PVEP examination was performed on two groups of kittens regularly every week. The successful establishment of amblyopia model was based on the standard that the amplitude of P100 wave in the right eye (covering eye) of the experimental group was lower than that of the other three groups, and the latency of P100 wave was higher than that of the other three groups^11–13.

At the age of 7 weeks, after PVEP detection, all kittens were euthanized by intraperitoneal injection of 2% pentobarbital sodium (100mg/kg) according to the American Veterinary Medical Association (AVMA) Animal Euthanasia Guidelines (2020). According to the Atlas of Feline Anatomy For Veterinarians, the left lateral geniculate body of kittens was isolated and fixed in 4% paraformaldehyde. The expression of ARC/Arg3.1 was detected by immunohistochemistry (IHC) and in situ hybridization (ISH), and the apoptosis of neurons in lateral geniculate body was detected by TdT-mediated dUTP Nick-End Labeling (TUNEL).

PVEP detection

All kittens were anesthetized intraperitoneally with 1% pentobarbital sodium (35mg/kg). The refractive errors of all kittens were detected by retinoscopy and corrected by lenses. Then, the animal electrode needles (RL-1223000030-RC-D, Roland Consult Stasche Finger Gmbh) were inserted into the middle of forehead, occipital and posterior part of ear tip of kittens. Place the kitten 40cm away from the vertical line in the center of the display screen, and adjust the head position so that its visual axis is perpendicular to the screen. Set the program of Reti-Port/Scan 21B (Roland Consult Stasche Finger Gmbh), select the chessboard flip mode, the time frequency 1Hz, 0.3cpd mode, a contrast of 97%, superimposed 64 times and sampling time of 300ms¹⁴. The test was repeated 3 times for each eye of each kitten and the average value was obtained.

Immunohistochemical

Paraffin sections were prepared and set the thickness of the slices to 4 µm. The citric acid (pH 6.0) was used for antigen retrieval. After endogenous peroxidase was blocked, serum blocking was performed. Then, the first antibody (ARC/Arg3.1) (Proteintech Group, Inc, China, 16290-1-AP), the second antibody and SABC were added in turn. DAB (Boster Biological Technology Co., Ltd., China, AR1022) was used for color development, and the positive reaction color of DAB was brown-yellow; the nucleus was blue after hematoxylin staining, and then dehydrated and sealed. Three fields of view were randomly selected for each slice for statistical analysis. Chemiluminescence was measured using image analysis (Image Pro Plus 6.0).

In situ hybridization

Paraffin sections were prepared and set the thickness of the slices to 4 µm. After endogenous peroxidase was blocked. Add pre-hybridization solution, hybridization solution, BSA, anti-digoxigenin-labeled peroxidase, and SABC (Boster Biological Technology Co., Ltd., China, MK1612) in sequence. The hybridization solution contains ARC/Arg3.1 mRNA probe (5`-CGCTG GGTCA AGCGT GAGAT GCACG TGTGG AGGGA-3`; 5`-TATTG GCTGT CCCAG ATCCA GAACC ACATG AATGG-3`; 5`-TGGCG TAAGC GGGAC CTGTA CCAGA CACTG TATGT-3`). DAB (Boster Biological Technology Co., Ltd., China, AR1022) was used for color development, and the positive reaction color of DAB was brown-yellow; the nucleus was blue after hematoxylin staining, and then dehydrated and sealed. Three fields of view were randomly selected for each slice for statistical analysis. Chemiluminescence was measured using image analysis (Image Pro Plus 6.0).

TUNEL

Paraffin sections were prepared and set the thickness of the slices to 4 µm. After digested with protease K, 3% hydrogen peroxide was added to block endogenous peroxidase. Add Labeling Buffer, 5% BSA, the Anti-DIG-Biotin and SABC (Boster Biological Technology Co., Ltd., China, MK1015) in sequence. DAB (Boster Biological Technology Co., Ltd., China, AR1022) was used for color development, and the color of DAB positive reaction was brown-yellow; the nucleus appeared blue after hematoxylin staining, and then dehydrated and sealed. Three fields of view were randomly selected for each slice for statistical analysis. Chemiluminescence was measured using image analysis (Image Pro Plus 6.0).

Statistical analysis

The statistical analysis software was Stata/SE 16.0 All data are expressed as means ± standard deviation (± s). One-way analysis of variance (LSD) was performed on the PVEP results of each eye in the experimental group and the control group. Paired sample t-test was used to compare the differences between different eyes in the same group, two independent sample t-test was used to compare the difference between the right eye in the control group and the experimental group. The results of IHC, ISH and TUNEL were tested by independent sample t-test. Pearson correlation coefficient analysis was performed on the IHC, ISH and TUNEL data of the control group and the experimental group.

P100 wave of PVEP

At the age of 3 weeks, there was no statistical difference in latency (F = 0.629, P = 0.601) and amplitude (F = 0.013, P = 0.998) of P100 wave between the right eye of the experimental group, the left eye of the experimental group, the right eye of the control group and the left eye of the control group. With the increase of age, the latency of four groups of P100 waves showed a downward trend, while the amplitude showed an upward trend. Moreover, although the latency and amplitude of P100 wave in the right eye of the experimental group also showed a changing trend, the overall change was lower than that of the other three groups.

At the age of 5 weeks, there were statistical differences in latency (F = 7.306, P = 0.001) and amplitude (F = 3.674, P = 0.021) of P100 wave among the four groups. The latency of P100 wave in the right eye of experimental group was higher than that in the left eye of experimental group (P = 0.006), the right eye of control group (P = 0.001) and the right eye of control group (P < 0.001). The amplitude of P100 wave in the right eye of experimental group was lower than that in the left eye of experimental group (P = 0.027), the right eye of control group (P = 0.006) and the right eye of control group (P = 0.009).

At the age of 6 weeks, there were statistical differences in latency (F = 8.593, P < 0.001) and amplitude (F = 4.467, P = 0.009) of P100 wave among the four groups. The latency of P100 wave in the right eye of experimental group was higher than that in the left eye of experimental group (P = 0.003), the right eye of control group (P < 0.001) and the right eye of control group (P < 0.001). The amplitude of P100 wave in the right eye of experimental group was lower than that in the left eye of experimental group (P = 0.009), the right eye of control group (P = 0.004) and the right eye of control group (P = 0.004).

At the age of 7 weeks, there were statistical differences in latency (F = 8.691, P < 0.001) and amplitude (F = 6.275, P = 0.002) of P100 wave among the four groups. The latency of P100 wave in the right eye of experimental group was higher than that in the left eye of experimental group (P = 0.001), the right eye of control group (P < 0.001) and the right eye of control group (P < 0.001). The amplitude of P100 wave in the right eye of experimental group was lower than that in the left eye of experimental group (P = 0.002), the right eye of control group (P = 0.001) and the right eye of control group (P = 0.001).

The results showed that after 5 weeks of age, monocular form deprivation amblyopia had formed in the right eye of kittens in the experimental group. (Fig. 1, Table 2–3). (relevant data is available at https://figshare.com/s/41efd7f80329308c0d0a)

Table 2

P100 latency in each group. (± S, ms)
Time (weeks)	right eye of the experimental group	left eye of the experimental group	right eye of the control group	left eye of the control group	F	P
3	118.66 ± 3.10	118.36 ± 4.11	117.04 ± 2.41	117.27 ± 2.85	0.629	0.601
4	113.00 ± 3.01	110.22 ± 3.52	108.84 ± 3.45*	109.50 ± 3.34*	3.005	0.043
5	109.22 ± 2.89	105.12 ± 2.04*	104.29 ± 2.52*	103.07 ± 4.48*	7.306	0.001
6	106.55 ± 3.90	101.03 ± 2.86*	99.11 ± 3.76*	99.08 ± 4.51*	8.593	< 0.001
7	104.80 ± 3.94	98.45 ± 3.82*	96.69 ± 4.20*	97.59 ± 3.83*	8.691	< 0.001
F	26.546	55.777	59.068	44.393
P	< 0.001	< 0.001	< 0.001	< 0.001
*Compared with the right eye of the deprivation group, there was a difference (P < 0.05).

Table 3

P100 amplitude in each group. (± S, V)
µ
Time (weeks)	right eye of the experimental group	left eye of the experimental group	right eye of the control group	left eye of the control group	F	P
3	4.995 ± 0.661	5.073 ± 0.854	5.027 ± 1.033	5.051 ± 1.081	0.013	0.998
4	6.413 ± 0.819	6.909 ± 1.066	7.274 ± 0.782*	7.274 ± 0.832*	2.139	0.112
5	7.138 ± 0.681	7.936 ± 0.805*	8.139 ± 0.820*	8.097 ± 0.775*	3.674	0.021
6	7.464 ± 0.907	8.730 ± 0.652*	8.897 ± 1.180*	8.880 ± 1.273*	4.467	0.009
7	7.687 ± 0.927	9.109 ± 0.908*	9.181 ± 0.996*	9.244 ± 0.941*	6.275	0.002
F	18.178	34.946	29.418	28.011
P	< 0.001	< 0.001	< 0.001	< 0.001
*Compared with the right eye of the experimental group, there was a difference (P < 0.05).

Immunohistochemical

The results of IHC showed that ARC/Arg3.1 protein was expressed in sections of the experimental group and the control group. ARC/Arg 3.1 protein expression in the cytoplasm, which was brown-yellow, and the nucleus was blue. At the age of 7 weeks, the average optical density of positive cells in the experimental group was lower than that in the control group (P < 0.001). And the number of positive cells in the experimental group was lower than that in the control group (P < 0.001). (Fig. 2, Table 4) (relevant data is available at https://figshare.com/s/41efd7f80329308c0d0a)

Table 4

ARC/Arg3.1 immunohistochemical results of visual cortex in each group. (± S)
Group	Mean optical density of positive	Positive cell number
Experimental group	0.009294 ± 0.002941	19.10 ± 7.60
Control group	0.017041 ± 0.004765	34.83 ± 11.64
t	-7.579	-6.199
P	< 0.001	< 0.001

In situ hybridization

The results of ISH showed that ARC/Arg3.1 mRNA was expressed in sections of the experimental group and the control group. ARC/Arg 3.1 mRNA expression in the cytoplasm, which was brown-yellow, and the nucleus was blue. At the age of 7 weeks, the average optical density of positive cells in the experimental group was lower than that in the control group (P < 0.001). And the number of positive cells in the experimental group was lower than that in the control group (P < 0.001). (Fig. 3, Table 5) (relevant data is available at https://figshare.com/s/41efd7f80329308c0d0a)

Table 5

ARC/Arg3.1 in situ hybridization results of visual cortex in each group. (± S)
Group	Mean optical density of positive	Positive cell number
Experimental group	0.006526 ± 0.001905	43.07 ± 11.52
Control group	0.013550 ± 0.004042	58.33 ± 14.53
t	-8.609	-4.508
P	< 0.001	< 0.001

TUNEL

The results of TUNEL showed that there were positive cells in the slices of the experimental group and the control group. The nucleus of positive cells was brown-yellow, while that of negative cells was blue. At the age of 7 weeks, the average optical density of positive cells in the experimental group was higher than that in the control group (P < 0.001). The number of positive cells in the experimental group was more than that in the control group (P < 0.001). (Fig. 4, Table 6) (relevant data is available at https://figshare.com/s/41efd7f80329308c0d0a)

Table 6

Tunel staining results in each group. (± S)
Group	Mean optical density of positive	Positive cell number
Experimental group	0.005364 ± 0.001867	45.73 ± 16.48
Control group	0.003595 ± 0.001586	27.07 ± 14.09
t	3.954	4.715
P	< 0.001	< 0.001

The analysis of correlation

Pearson correlation coefficient indicated that the intensity of TUNEL positive reaction was negatively correlated with mean optical density of positive cells from ARC/Arg3.1 protein (PCCs=-0.415, P = 0.001) and mRNA (PCCs=-0.409, P = 0.001) expression at the age of 7 weeks, and ARC/Arg3.1 protein expression was positively correlated with ARC/Arg3.1 mRNA expression (PCCs = 0.958, P < 0.001). (Table 7).

Table 7

The analysis of correlation analysis among ISH, IHC and TUNEL. (PCCs, P)
Group	IHC	ISH	Tunel
Group	IHC	ISH	Tunel
IHC	-	0.958, < 0.001	-0.415, 0.001
ISH	0.958, < 0.001	-	-0.409, 0.001
Tunel	-0.415, 0.001	-0.409, 0.001	-

Among the animals commonly used to establish amblyopia models, the common species are mice, rats, monkeys and cats¹⁵. The application of kittens as a model of form deprivation amblyopia can be traced back to 1971. Eysel et al.¹⁶ established an amblyopia kitten model by form deprivation, and discussed the changes of medial and lateral geniculate bodies during form deprivation. For kittens, the effects of form deprivation on vision usually peaks from birth to 4 weeks old. After 12 weeks of age, the form deprivation caused by occlusion can hardly interfere with their visual development^{17, 18}. Therefore, in this study, 3-week-old kittens were selected, which can fully ensure that their covering eyes are affected by form deprivation factors during the visual development period of kittens, and have maximum interference on their visual development, so as to successfully establish a monocular form deprivation amblyopia kitten model. Some studies have shown that when the eyes of kittens are covered by monocular deprivation, the transformation of ocular dominance column can occur in a short time^{19, 20}. And with the prolongation of deprivation time, the deviation of dominant column pairs will become more obvious.

In order to observe the form deprivation eyes of kittens and compare whether form deprivation amblyopia is formed. In this study, the results of PVEP detection were used as the basis for the establishment of amblyopia model. At present, it has been mature and applied to the establishment of amblyopia animal model²¹. The retina is stimulated by external light, which will produce potential changes and form nerve impulses. Nerve impulses pass through optic nerve, optic chiasma, optic bundle, lateral geniculate body and optic radiation in turn, and finally pass to the visual center of cerebral cortex. Under the condition of normal visual development of kittens, the conduction velocity and mode of cells related to vision can stimulate consistent potential activity, and form harmonious synchronous oscillation in front of and in visual cortex, and then produce regular waveform²².

The results showed that with the development of visual system of kittens, after 4 weeks of covering, the latency of P100 wave decreased and the amplitude increased in both eyes of the experimental group and control group. However, the experimental group of right eye compared with the other three groups, although there is a certain degree of changes, but the overall range is still lower than the other three groups. Moreover, after 2 weeks of occlusion (at the fifth week of age), the latency of P100 wave in the occluded eyes of the experimental group was significantly higher than that of the other three groups, and the amplitude of P100 wave was significantly lower than that of the other three groups. Therefore, we believe that form deprivation amblyopia developed in the right eye of the experimental group at the fifth week of age. This result is consistent with the results of previous studies^{23, 24}.

The balance of excitation and depression at the axonal level of visual cortex is the condition of maintaining the normal development and function of visual cortex, and it is also an important factor affecting the plasticity of visual system²⁵. The first synaptic replacement of optic nerve impulse in brain occurs in lateral geniculate body, and each lateral geniculate body receives projections from temporal optic nerve fibers of ipsilateral retina and nasal optic nerve fibers of contralateral retina, and this projection has strict local regional correspondence. As a semi-crossover animal, the optic nerve fibers of kittens pass through the optic chiasma to the lateral geniculate body, and the lateral geniculate body of kittens can be divided into A, A1, C and C1-C3 layers. Layers A, C and C2 received projections from contralateral ocular fibers, while layers A1 and C1 received projections from ipsilateral ocular fibers. Some studies have found that C-fos, GABA and brain-derived neurotrophic factor (BDNF) in the contralateral lateral geniculate body of the amblyopic eyes are down-regulated compared with those in the ipsilateral amblyopic eyes in kittens with monocular form deprivation amblyopic eyes^26–28. Therefore, this is also the basis for our study of form deprivation amblyopia by using the contralateral lateral geniculate body of model eyes.

Synapse, as the structural basis of information transmission between neurons, is the key part of visual development plasticity. Some studies have suggested that synaptic plasticity is considered as the most critical link in the pathogenesis of amblyopia²⁹. The neural storage in neural networks is considered to depend partly on the plasticity of synapses^{30, 31}. Synaptic plasticity refers to the change of synaptic connections between neurons when synapses are in use or disuse^{31, 32}. In terms of time, it can be divided into two types: LTP and LTD³³. LTP is a kind of information storage mode at synaptic level, and the induction mechanism of LTP is mainly Ca2⁺ influx caused by the change of NMDAR channel in postsynaptic membrane, which triggers a series of biochemical processes in cells. LTD refers to activity-dependent persistent potential attenuation induced on an unstimulated pathway, which is the opposite of LTP. For example, the visual cortex response caused by visual deprivation is an LTD phenomenon.

Considering the induction and expression mechanism of LTP and LTD in the lateral geniculate body, it is similar to hippocampus^34–38. For example, NMDAR needs to be activated, which leads to the activation of a series of intracellular kinases and the redistribution of AMPA receptors. The first change in the plasticity of lateral geniculate body is synaptic efficiency, which does not need to synthesize new proteins, and then there will be long-term changes in neural pathways. However, long-term changes in neural pathways require gene expression and new protein synthesis, so kinase activation will definitely lead to gene expression, which may be realized through the activation of transcription factors.

Arc/arg3.1 encodes a protein of about 400 amino acids, which has no catalytic or other known functional motifs^39–41. Arc/Arg3.1 protein interacts directly or indirectly with many proteins, indicating that it has the function of pivotal protein^42–48. Most of its functions are thought to occur at postsynaptic sites. Biochemical and electron microscopic studies show that Arc/Arg3.1 protein exists in the postsynaptic density of activated neurons^49–51.

As an archetypal immediate-early genes, ARC/Arg3.1 is generally considered as a reliable marker of neuronal activity^{52, 53}. ARC/Arg3.1 is also necessary for various forms of learning and memory, and some studies suggest that it is related to synaptic plasticity⁴¹. For example, ARC/Arg3.1 could regulate the expression of AMPAR during homeostatic plasticity with LTD and then maintain LTP^54–56.

ARC/Arg3.1 plays a key role in the long-term synaptic plasticity of excitatory synapses and in memory and postnatal cortical development⁵⁷. It is found that LTP and LTD, heterosynaptic LTD (inverse synaptic tagging) and homeostatic scaling all require the synthesis of ARC/Arc3.1, but the specific mechanism that determines the prominent changes is still unclear⁵⁷. When BDNF is injected into hippocampus, a transcription-dependent LTP induced by ARC/Arg3.1 mRNA in granulosa cell body and dendrite can be induced^{58, 59}. Moreover, the expression of BDNF is associated with the maintenance of high-frequency stimulation long-term potential (HFS-LTP)⁵⁶. Injection of Arc antisense oligodeoxynucleotides (Arc-AS) before injection of BDNF inhibits the induction of LTP, which indicates that the process is completely downstream of BDNF signaling pathway^{56, 60}. After injecting BDNF for 2 hours, the LTP can be restored to the baseline level by injecting Arc-AS again. However, after injecting BDNF for 4 hours, Arc-AS injection has no effect⁵⁶. Therefore, some researchers believe that the newly synthesized ARC/Arg 3.1 can regulate the expression, expression and consolidation of LTP induced by HFS-LTP and exogenous BDNF⁶¹. In addition, Qi et al.⁶² showed that ARC/Arg3.1 can be activated and up-regulated by PKA/CREB and ERK/CREB signaling pathways and they found a significant increase in the number of neuronal apoptosis in the model group after ARC/Arg 3.1 gene knockout.

In this study, the apoptosis of neurons in lateral geniculate body and the expression of ARC/Arg3.1 gene were compared between two groups of kittens, which proved that there was a certain correlation between the expression of ARC/Arg3.1 gene and amblyopia and neuronal apoptosis. Based on these results, we speculate that the expression of ARC/Arg3.1 in the contralateral lateral geniculate body of the amblyopic cats was down-regulated by form deprivation factors, which further led to changes in the function and apoptosis of neurons in the lateral geniculate body.

It is particularly noteworthy that there is a certain relationship between the expression of BDNF and ARC/Arg3.1. Then, whether the intervention of ARC/Arg3.1 expression in amblyopia kittens will change the expression of BDNF in lateral geniculate body and further improve visual function. This is worthy of further study.

To sum up, the expression of ARC/Arg3.1 is associated with monocular form deprivation amblyopia and apoptosis of lateral geniculate body cells. This study speculates that ARC/Arg3.1 gene plays an important role in visual development.

ARC/Arg3.1

activity-regulated cytoskeleton-associated

LTP

long-term potentiation

LTD

long-term depression

PVEP

Pattern visual evoked potential

IHC

immunohistochemistry

ISH

in situ hybridization

TUNEL

TdT-mediated dUTP nick-end labeling

HFS-LTP

high-frequency stimulation long-term potential

Arc-AS

Arc antisense oligodeoxynucleotides

Acknowledgements

Not applicable.

Authors` contributions

Yunchun Zou participated in the design of the experiment, analysed the data, and modified the manuscript. Haobo Fan and Ying Wang contributed equally to this work and participated in creating the animal model, specimen collection, experimental manipulation, data collection and analysis, and manuscript writing. Weiqi Song, Juan Xie Siyu Chen, and Xiuping Tang participated in creating the animal model, PVEP testing, specimen collection and experimental operation. All authors read and approved the final manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article Natural Science Foundation of Sichuan (No.2022NSFSC0754). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Or all relevant datasets related to the study can be found in the specified (database.https://figshare.com/s/41efd7f80329308c0d0a).

Ethics approval and consent to participate

The study that has been performed according to the ARRIVE guidelines was approved by the Medical Ethics Committee of North Sichuan Medical College and supervised throughout the process. (NSMC Appl. No. 2021 [66])

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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ARC/Arg3.1 expression in the lateral geniculate body of monocular form deprivation amblyopic kittens

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Background

Methods

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Animals model establishment

PVEP detection

Immunohistochemical

In situ hybridization

TUNEL

Statistical analysis

Results

P100 wave of PVEP

Immunohistochemical

In situ hybridization

TUNEL

The analysis of correlation

Discussion

Conclusions

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