Dynamic changes of FasL protein expression in retinal tissue with aging in retinal degeneration mice / 中华实验眼科杂志

Background Retinal transplantation is a new approach to the treatment of retinal degeneration diseases,and how to avoid or reduce the immune rejection after transplantation is a problem.In experimental studies on retinal transplantation,C57BL/6 mice are often used as donors and rd mice serve as recipients.Studies showed that Fas ligand (FasL) protein induces the apoptosis of Fas+ inflammatory cells by FasL/Fas signal pathway,speculating that FasL protein is associated with immune rejection after transplantation.Objective The aim of this study was to investigate FasL protein expression change in retinas of C57BL/6 mice and rd mice with aging and reveal the immune characteristics of the mouse retinas.Methods The frozen sections of eyeball from C57BL/6 mice and rd mice at the age of postnatal (PN)-0 week,PN-1 week,PN-2 week,PN-3 week and PN-4 week were prepared.The expression of FasL protein in the mouse retinas was examined by immnofluorescense technique.Images were acquired by fluorescence microscope and analyzed semi-quantitively by software from laser scanning confocal microscope as the fluorescence intensity (FI).The results were compared among different strains of mice.Results The retina developed imperfectly in PN-1 week C57BL/6 mice and FasL protein was positively expressed in the whole retina.In PN-2,3 and 4 week C57BL/6 mice,retinas finished the development with 10 layers,and retinal pigment epithelium (RPE),inner segment (IS),outer limiting membrane (OLM),outer plexiform layer (OPL),inner nuclear layer (INL),inner plexiform layer (IPL) and ganglion cell layer (GCL).Retinal structure and expression of FasL protein in the whole retina of rd mice in the PN-1 week were similar with C57BL/6 mice,however,ONL cells of rd mice were evidently decreased with aging.The RPE,OPL,INL,IPL and GCL expressed the FasL protein in PN-2,PN-3 and PN-4 week rd mice.The mean FI of FasL protein in the RPE layer was 184.199±16.747,186.797±7.904 and 184.319± 18.795 in rd mice of PN-2 week,PN-3 week and PN-4 week,which were significantly higher than 160.402±22.851,160.995 ±22.799 and 105.787 ± 17.676 in C57BL/6 mice (t =-3.360,P =0.002;t'=-4.277,P =0.000;t =-12.175,P=0.000).There were not significant differences in the mean FI of FasL protein in IPL between C57BL/6 mice and rd mice at ages of PN-2 week,PN-3 week and PN-4 week (all at P＞0.05).Conclusions The cells of retinal ONL are gradually decreased with the development of rd mice,and FasL protein expression intensity in RPE is evidently enhanced in rd mice compared with C57BL/6 mice.

Effect of maternal separation stress on behavior of neonatal rd mice / 中国比较医学杂志

Objective To evaluate the effect of maternal separation stress on the behavior of neonatal rd mice.Methods Neonatal rd mice were divided into maternal separation (MS) group (n=9) and control group (n=9).MS-stress was induced in the MS group by 4-hour-separation per day for 28 days.Open field test,elevated plus maze test,forced swim test and tail suspension test were used to evaluate the anxiety-like and depression-like behavior of the neonatal rd mice.Results The stay time and distance travelled of MS group in the central zone were 0.88％ and 28.17±5.65 cm,respectively,significantly shorter than that of the control group (2.61％,109.9±9.79 cm.P =0.04,P =0.001).Compared with the control group,the stay time in open arms of the MS group was significantly decreased (P<0.01),while the immobility time in forced swim test and tail suspension test of the MS group were 126.5±10.22 s and 21.56±6.83 s,significantly longer than that of the control group (77.75±16.83 s,P =0.02,7.37±3.22 s,P =0.03).Conclutions The 28-day maternal separation stress can significantly increase the anxiety-like and depression-like behavior in neonatal rd mice.

Inhibitor of NADPH oxidase slow photoreceptor cell death in the retinal degeneration of rd mice / 中华实验眼科杂志

Background Our previous study demonstrated that microglial activation is closely associated with photoreceptor apoptosis in rd mice.Recent studies on central nervous system (CNS) showed that activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the microglia activation and neural cell death.However,the mechanism of NADPH oxidase during the retinal degeneration and the effect of NADPH oxidase inhibitor on photoreceptor apoptosis are concerned.Objective The aim of this study was to further explore the production of reactive oxygen species (ROS) by NADPH oxidase in the retinal degenerative process of rd mice and protection of NADPH oxidase inhibitor on photoreceptors.Methods Sixty rd mice at postnatal day 9 (P9) were randomized into the experimental group and the control group by throwing coins method.Apocynin,a NADPH oxidase inhibitor,was intraperitoneally injected in the dose of 10 mg/kg (0.01 ml/kg) once daily for 5 days (P13) in the experimental group,and the equal amount of PBS was used in the same way in the control group,and 10 C57BL/6N mice without injection of any drugs served as the wild type mice group.All the mice were sacrificed in P14 for the preparation of retinal sections.The expression of ROS in the retina was detected by dihydroethidium (DHE) fluorescence staining.Expression level of rhodopsin mRNA in the photoreceptor of the mice was determined by real-time PCR,and the thickness of retinal outer nuclear layer (ONL) in the mice of the experimental group and the control group was measured using hematoxylin & eosin staining.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology.Results DHE staining showed that the ROS presented with the red fluorescence in the mouse retinas.In the rd mice of the experimental group,the ROS fluorescence intensity was dramatically enhanced in comparison with C57BL/6N mice,but weakened in comparison with the rd mice of the control group.Real-time PCR revealed that the relative expressing level of rhodopsin mRNA in the photoreceptor was (4.21±0.33) in the experimental group and (0.93±0.24) in the control group,showing a significant difference between them (t =2.360,P =0.000).The thickness value of retinal ONL was (35.95±1.63)μm in the mice of the experimental group,which was significantly higher than that in the mice of the control group ([23.17±1.38] μm) (t=3.850,P=0.016).Conclusions In the retinal degeneration of rd mice,activation of NADPH oxidase increases the ROS production.Apocynin can slow the apoptosis procedure of photoreceptor cells of rd mice.

Delay of Photoreceptor Cell Degeneration in rd Mice by Systemically Administered Phenyl-N-tert-butylnitrone

PURPOSE: To study the effect of systemic administration of phenyl-N-tert-butylnitrone (PBN) on the degeneration of photoreceptor cells in rd mice. METHODS: PBN was injected intraperitoneally into FVB/rd mice on postnatal days (P) 5 to 14 (group A), and P10 to 18 (group B). At days P14, 16, 18, 20 and 27, morphological changes and apoptosis were analyzed by staining with hematoxylin and eosin or DAPI. The effect of PBN on apoptosis was analyzed in retinal pigment epithelial (RPE) cells by the measurement of caspase-3 activity. RESULTS: In control and group B mice, the outer nuclear layer (ONL) of the retina was composed of 8-10 rows at P12, and rapidly decreased to one row at P18. In group A mice, the ONL was preserved with 5-7 rows at P18, and decreased to one row at P22. PBN inhibited caspase-3 activity in cultured RPE cells. CONCLUSIONS: PBN delayed, but did not block, the degeneration of photoreceptor cells in rd mice. PBN may exert its inhibitory effect during the early phase of photoreceptor cell degeneration.

HEREDITARY RETINAL DYSTROPHY OF rd MICE AND PHOTORECEPTORS APOPTOSIS / 解剖学报

Objective To study the development of hereditary dystrophic retina of rd mice and photoreceptors apoptosis. Methods Retinal sections of rd mice and their controls at different ages ranging from postnatal days 5 to 40 were examined by morphological(light and electronic microscope), morphometric and TUNEL analysis. Results Compared with age-matched control mice, the retinal dystrophy of rd mice began at postnatal days 10, resulting in rapid loss of photoreceptors and reaching a peak at postnatal days 18. TUNEL postitive nucleus of photoreceptor cells emerged from postnatal days 10 and reached a peak at postnatal days 14 and 16. Ultrastructure of photoreceptor cell layer showed marked nuclear pyknotosis and chromatin margination. Apoptotic bodies of photoreceptor cells were observed.Conclusion Rd mice retina degenerated during the process of maturity. Photoreceptor cell death occurred through apoptosis.