ABSTRACT
Objective:
To study the differential expression profiling of the transcripts modified by m5C
methylation in a
rat model of
N-methyl-D-aspartate (
NMDA)-induced
retinal excitotoxicity.
Methods:
A total of 65 Sprague Dawley
male rats aged 7-8 weeks were randomly divided into two groups normal
control group and
NMDA group. The right
eye (model
eye) of
rats in the
NMDA group were injected with 50.0 mmol/L of
NMDA 3 μl in the vitreous cavity, while in the normal
control group, equal volume of
normal saline was injected into the vitreous cavity. After 1 week of the
injection, the
optic nerve conduction function of
rats was detected by
visual evoked potential. The whole structure of
rat retina was observed by
hematoxylin-
eosin staining, and the thickness of each
retinal layer and the number of
retinal ganglion cell layer were detected. The number of β3
tubulin immunofluorescence positive
cells was detected by
immunofluorescence staining on
retinal stretched preparation. Total
RNA was extracted from the retinas of normal
control group and
NMDA group, and high-throughput m5C modified
RNA was sequenced, and
bioinformatics analysis was performed. The relative expression levels of SLFN3, PLXNB3, CD36 and HIC2
mRNA in
retina were detected by real-
time quantitative
polymerase chain reaction. The comparison between the two groups was performed using an unpaired t test.
Results:
The P1 latency of
control group and
NMDA group were (117.86±6.48) and (148.46±3.78) ms, and the amplitudes were (42.57±2.41) and (8.68±0.63) μV, respectively. Compared with the normal
control group, the latency period was prolonged and the amplitude was significantly decreased in the
NMDA group, with statistical significance ( P<0.001). In normal
control group,
retinal ganglion cells (RGC) were uniformly arranged with large round nuclei. In
NMDA group, the volume of
retinal RGC was atrophied and the number of RGC was reduced. The total
retinal thickness in the
control group and
NMDA group was (207.51±12.76) μm and (187.51±12.54) μm, respectively. The number of β3
tubulin positive
cells was 79.86±6.56 and 29.36±2.16, respectively. Compared with normal
control group, the total
retinal thickness and the number of β3
tubulin positive
cells in
NMDA group were decreased, with statistical significance ( P <0.001). Compared with the
control group, 576 differentially expressed m5C
mRNA were screened in the
NMDA group, among which 230 up-regulated and 346 down-regulated
genes were detected, respectively. The results of
biological information
analysis showed that compared with the
control group, the upregulated m5C
mRNA in the
NMDA group was mainly involved in
biological processes such as
perception and
cell-
cell adhesion, and was mainly concentrated in the
cytokine-
cytokine receptor interaction and neural active
ligand-receptor interaction pathway. The
biological processes in which down-regulated m5C
mRNA was mainly involved in
biological processes such as
G-protein-coupled receptor signaling pathway and
cell communication, which were mainly concentrated in primary immune deficiency pathway and neural active
ligand-receptor interaction pathway. Real-
time quantitative
polymerase chain reaction detection results showed that compared with the normal
control group, the relative expression levels of SLFN3 and PLXNB3
mRNA in the
retina of
rats in
NMDA group were significantly increased, while the relative expression levels of CD36 and HIC2
mRNA were significantly decreased, with statistical significance ( P<0.05).
Conclusion:
In
NMDA induced
retinal excitatory
toxicity rat models, m5C modified
retinal transcriptome showed abnormal expression.
