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ABSTRACT Purpose: The study aimed to investigate the correlation between arterial hemodynamics measured by color Doppler ultrasonography and retinal microarchitecture parameters determined by spectral-domain optical coherence tomography (SD-OCT) in pseudoexfoliation glaucoma. Methods: This prospective study included 82 participants. Peripapillary retinal nerve fiber layer, ganglion cell inner plexiform layer, and ganglion cell complex values were measured. Ophthalmic artery and central retinal artery flows were evaluated with color Doppler ultrasonography, and resistivity index values were calculated. Results: The study included 47 controls and 35 pseudoexfoliation glaucoma cases. In pseudoexfoliation glaucoma group, mean peripapillary retinal nerve fiber layer and ganglion cell complex thickness were statistically significantly lower in all quadrants compared to controls (p<0.001). Resistivity index values of the ophthalmic and central retinal arteries were significantly higher in pseudoexfoliation glaucoma group than in the controls (p<0.001 and r=0.684). Resistivity index values of the ophthalmic and central retinal arteries with ganglion cell complex thickness correlated significantly. On the other hand, no significant relationship for retinal nerve fiber layer thickness was identified. Conclusions: Structural changes (ganglion cell complex and ganglion cell inner plexiform layer) in patients with pseudoexfoliation glaucoma and early glaucomatous loss showed a significant correlation with changes in ocular vascular hemodynamics. In cases where systemic vascular resistance is increased, ganglion cell complex and ganglion cell inner plexiform layer may not exactly reflect glaucoma state. In such cases, thickness changes in the retinal nerve fiber layer may give more realistic results regarding glaucoma. We have seen that pseudoexfoliation glaucoma-induced structural deterioration and increased resistance in ocular hemodynamics correlated with ganglion cell complex, but not retinal nerve fiber layer.
RESUMO Objetivo: Investigar a correlação entre a hemodinâmica arterial, medida pela ultrassonografia com Doppler colorido, e os parâmetros de microarquitetura da retina, determinados pela tomografia de coerência óptica de domínio espectral (SD-OCT) no glaucoma pseudoexfoliativo. Métodos: Foram incluídos 82 participantes neste estudo prospectivo. Foram medidos os valores da camada de fibras nervosas da retina peripapilar, da camada plexiforme interna de células ganglionares e do complexo de células ganglionares. Os fluxos da artéria oftálmica e da artéria central da retina foram avaliados com ultrassonografia por Doppler colorida e foram calculados os valores do índice de resistividade. Resultados: Foram incluídos no estudo 47 casos de controle e 35 casos de glaucoma pseudoexfoliativo. No grupo com glaucoma pseudoexfoliativo, a média da camada de fibras nervosas da retina peripapilar e a espessura do complexo de células ganglionares foram menores em todos os quadrantes em comparação com os controles, com significância estatística (p<0,001). Os valores do índice de resistividade das artérias oftálmica e central da retina foram significativamente maiores no grupo com glaucoma pseudoexfoliativo que nos controles (p<0,001 e r=0,684). Ao se compararem os valores do índice de resistividade das artérias oftálmica e central da retina com a espessura do complexo de células ganglionares, foi encontrada uma correlação significativa entre elas. Por outro lado, não detectamos uma relação significativa para a espessura da camada de fibras nervosas da retina. Conclusões: Alterações estruturais (complexo de células ganglionares, camada plexiforme interna de células ganglionares) em pacientes com glaucoma pseudoexfoliativo com perda glaucomatosa precoce mostraram uma correlação significativa com alterações na hemodinâmica vascular ocular. Nos casos em que a resistência vascular sistêmica é aumentada, o complexo de células ganglionares e a camada plexiforme interna de células ganglionares podem não refletir exatamente o estado do glaucoma. Nesses casos, alterações na espessura da camada de fibras nervosas da retina podem dar resultados mais realistas em relação ao glaucoma. Observou-se uma correlação da deterioração estrutural induzida pelo glaucoma pseudoexfoliativo e do aumento da resistência na hemodinâmica ocular com o complexo de células ganglionares, mas não com a camada de fibras nervosas da retina.
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In addition to visual field defects, occipital lobe injury can also cause fundus changes, such as retinal nerve fiber layer atrophy, ganglion cell complex atrophy and even optic nerve atrophy, and these fundus changes have a good correlation with the visual field defect site. It is considered to be caused by transneuronal retrograde degeneration (TRD) of retinal ganglion cells secondary to occipital lobe injury. These changes can be detected by means of optical coherence tomography, fundus examination, magnetic resonance imaging, etc. Among them, optical coherence tomography is more sensitive than other examinations. Here, the anatomical basis of TRD, case reports, pathogenesis, auxiliary examination, treatment and prognosis of TRD secondary to occipital lobe injury are reviewd.
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AIM:To investigate the effects and mechanisms of curcumin on apoptosis of retinal ganglion cells(RGCs)in chronic ocular hypertension rats.METHODS:A total of 21 Spraque-Dawley(SD)rats were randomly divided into 3 groups with 7 rats in each group. The rat models of chronic ocular hypertension were established by cauterization of the superior scleral veins in the high intraocular pressure model group and the curcumin treatment group, and the sham operation group only cut the conjunctiva without the cauterization of the superior scleral veins; the rats in the curcumin treatment group were intragastrically treated with curcumin at a dose of 4mL/kg, and the rats in the sham operation group and the high intraocular pressure model group were treated with pure water at a dose of 4mL/kg for 3wk. After 3wk, HE staining was used to observe the morphological and pathological changes of retina, the number of RGCs and the thickness of ganglion cell layer(GCL)in each group of rats; TUNEL staining was used to observe the apoptosis of RGCs and retinal cells in each group of rats; the expression levels of glutamate-cysteine ligase modifier subunit(GCLM)and heme oxygenase-1(HO-1)in the retina of each group of rats were detected by real-time fluorescence quantitative PCR, immunohistochemical staining and Western blot.RESULTS:Compared with the sham operation group, the retinal morphology of rats in the high intraocular pressure model group and the curcumin treatment group was disorganized, the number of RGCs was reduced, the GCL was thinner, the apoptosis rate of RGCs and retinal cells increased, and the expression levels of GCLM and HO-1 increased. Compared with the high intraocular pressure model group, the retinal morphology of rats in the curcumin treatment group was basically normal, the number of RGCs increased, the GCL thickened, the apoptosis rate of RGCs and retinal cells decreased, and the expression levels of GCLM and HO-1 increased.CONCLUSION:Curcumin can inhibit the apoptosis of RGCs in the rat model of chronic ocular hypertension by up-regulating the expression of antioxidant genes GCLM and HO-1.
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Objective:To observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro.Methods:New Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method.Results:The survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11±0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant ( P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant ( P<0.001); the difference between ET medium dose group and ET high dose group was statistically significant ( P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant ( P<0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group ( P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant ( P<0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant ( P=0.007). Conclusion:ET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.
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Retinal ganglion cells (RGCs) are final output neurons from the retina to the brain, which can transmit light signals and participate in image-forming vision (IFV) (image formation) and non-image-forming vision (NIFV) (non-image formation). Visual processing system not only transmits visual information of images, but also influences human physiological activities and behaviors by incoming optical signals, which is called NIFV.NIFV relies less on signals generated by conventional photoreceptor cells, but a special class of intrinsically photosensitive retinal ganglion cells (ipRGCs). ipRGCs are a subset of retinal ganglion cells that express melanopsin.The axons of the ipRGCs project to unique targets and modulate a broad range of NIFV behaviors, from basic physiological regulation (such as heart rate and pupil size) to more complex behavioral regulation (such as circadian rhythm) and even higher-level cognitive processes (such as anxiety and other emotions). NIFV circuit is an important response to light, and ipRGCs plays a vital role in NIFV circuit.This article reviewed the regulation of NIFV circuit in physiological activities and behaviors, summarized the relationship between the projections of ipRGCs to the NIFV function, and provided ophthalmologists with more knowledge of visual system.
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Objective:To investigate the effect and mechanism of NOD-like receptor family pyrin domain containing 12 (NLRP12) knockdown on inflammatory factor levels and retinal injury in retinal ganglion cells (RGCs) of rats with high intraocular pressure.Methods:Seventy SPF adult male SD rats were selected and randomized into control group, high intraocular pressure (IOP) group, high IOP+ small interfering RNA negative control (siNC) group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ recombinant rat caspase-1 (rrcaspase-1) group, with 14 rats in each group.Rats in the control group were only treated with conjunctival incision in the right eye, and ocular hypertension model was established in the other four groups with external scleral vein cauterization.High IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group were injected with siNC, siNLRP12 and siNLRP12+ rrcaspase-1 reagent via the tail vein, respectively.The IOP of the right eye was measured at 1 day, 1, 2 and 3 weeks after the operation.Three weeks after the operation, the retinal structure was observed by hematoxylin-eosin staining, and the number of RGCs in each group was counted.RGCs were divided into control group, rrcaspase-1 group, siNC+ rrcaspase-1 group, siNLRP12+ rrcaspase-1 group.The cells in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group were treated with rrcaspase-1, siNC+ rrcaspase-1 and siNLRP12+ rrcaspase-1 reagent for 24 hours, respectively.No treatment was given to the control group.The expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins in RGCs and retinal tissue were detected by Western blot.The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rat serum or cell culture supernatant were detected by enzyme-linked immunosorbent assay.The study protocol was approved by the Animal Ethics Committee of the First People's Hospital of Chenzhou (No.2020086).Results:Compared with control group, the IOP was higher in high IOP group at 1, 2 and 3 weeks after cauterization, and the differences were statistically significant (all at P<0.05). The retinal tissue was clear with the RGCs in a single layer arrangement in the control group.In the high IOP group and the high IOP+ siNC group, the RGCs layer was loose and the inner plexiform layer was thin.The inner plexiform layer was thickened in high IOP+ siNLRP12 group compared with high IOP group, and the RGCs layer was loose in the high IOP+ siNLRP12 group and the high IOP+ siNLRP12+ rrcaspase-1 group.The number of RGCs in control group, high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group was 119.31±23.25, 89.19±16.98, 88.87±13.92, 109.33±10.25 and 92.89±12.58, respectively, showing a statistically significant overall difference ( F=201.932, P<0.001). The number of RGCs was lower in the high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than the control group, higher in the high IOP+ siNLRP12 group than the high IOP+ siNC group, and lower in the high IOP+ siNLRP12+ rrcaspase-1 group than the high IOP+ siNLRP12 group, and the differences were statistically significant (all at P<0.05). The relative expressions of caspase-1 and cleaved-caspase-1 proteins and the concentrations of TNF-α and IL-1β in the retinal tissue were higher in high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than control group, higher in high IOP+ siNLRP12 group than high IOP+ siNC group, and higher in high IOP+ siNLRP12+ rrcaspase-1 group than high IOP+ siNLRP12 group (all at P<0.05). Relative expression levels of caspase-1 and cleaved-caspase-1 protein were increased in rrcaspase-1 group and siNC+ rrcaspase-1 group compared with control group, and relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 protein were decreased in siNLRP12+ rrcaspase-1 group compared with control group (all at P<0.05). The relative mass concentrations of TNF-α and IL-1β were increased in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group compared with the control group (all at P<0.05). Relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins and relative mass concentrations of TNF-α and IL-1β in siNLRP12+ rrcaspase-1 group were lower than those in siNC+ rrcaspase-1 group (all at P<0.05). Conclusions:Knockdown of NLRP12 can reduce the inflammatory response and retinal injury induced by high IOP by inhibiting the activation of caspase-1.
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Maintaining the homeostasis of intracellular components degradation and recycling, autophagy is a critical control mechanism of cellular quality. It promotes the degradation of cell components to provide nutrients and energy for cellular metabolism in stress response. The retina is a light-sensitive tissue that transduces and processes visual images in the eye, and it has a high demand for substances and energy. Basal autophagy is essential for holding retinal homeostasis and the normal function of the visual system. Therefore, the latest studies that investigating the participation of autophagy in eye diseases such as glaucoma, age-related macular degeneration, diabetic retinopathy, retinal dystrophies, and retinal detachment were summarized, providing a theoretical basis for the future treatment of eye diseases by regulating autophagy.
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A brand-new class of photoreceptors has been identified in the past 20a: intrinsically photosensitive retinal ganglion cells(ipRGC). With melanopsin as its photopigment, ipRGCs transmit light signals to non-imaging brain regions like the suprachiasmatic nucleus(SCN)and the olivary pretectal nucleus(OPN)to regulate circadian photoentrainment and pupillary light reflex; a small portion of the signals are projected to brain imaging regions like the dorsal lateral geniculate nucleus(dLGN)and superior colliculus(SC), to participate in imaging vision. There are six different ipRGC subtypes(M1～M6), each with its own morphological and physiological characteristics. In addition to receiving signaling inputs from the rods and cones, ipRGCs also regulate retinal signals through chemical and electrical synapses and play important roles in visual signaling and visual development. It has been discovered that ipRGCs are implicated in several systemic and ocular illnesses. Overall, various aspects of ipRGC are reviewed including the discovery, general physiological properties, signaling, and the relationship with disease in this work.
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Hallmarks of the pathophysiology of glaucoma are oxidative stress and apoptotic death of retinal ganglion cells (RGCs). Ginkgo biloba extract (EGb) with multi-target, multi-pathway functions has been reported to exert positive pharmacological effects on oxidative stress and damaged RGCs. However, the ingredients and anti-apoptotic targets of EGb in the treatment of open-angle glaucoma (OAG) have not been fully elucidated. Therefore, in-depth analysis is necessary for further research. Ginkgo biloba-related and anti-apoptotic targets were identified and then combined to obtain the intersection, representing the potential anti-apoptotic targets of Ginkgo biloba. In addition, compound-anti-apoptotic target and OAG-target protein-protein interaction network were merged to obtain five core genes and compound-OAG-anti-apoptotic target protein-protein interaction network. Consequently, the active compounds and anti-apoptotic targets of Ginkgo biloba in the treatment of OAG were identified, namely luteolin, β-sitosterol, kaempferol, stigmasterol, quercetin, and p53, Bax, Bcl-2, Caspase-3 and Caspase-9, respectively. For the anti-apoptotic targets of Ginkgo biloba in the treatment of OAG, Gene Ontology (GO) and pathway analysis were executed to confirm the gene functions of Ginkgo biloba in antagonizing apoptosis of RGCs. The pathway enrichment was mainly involved in transcriptional activation of p53 responsive genes, activation of caspases and apoptotic processes. Finally, we confirmed the results of the network analysis by H2O2 treated RGC-5 cells in vitro. The results demonstrated that EGb protection can effectively diminish H2O2-induced apoptosis by inhibiting p53 acetylation, reducing the ratio of Bax/Bcl-2 and suppressing the expression of specific cleavage of Caspase-9 and Caspase-3.
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Humans , Ginkgo biloba , Glaucoma, Open-Angle , Hydrogen Peroxide , Network Pharmacology , Plant Extracts , Retinal Ganglion CellsABSTRACT
Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.
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Transsynaptic retrograde degeneration of optic neuropathy (TRDON) refers to the degeneration and/or apoptosis of presynaptic neurons (retinal ganglion cells) caused by damage to the lateral geniculate body and post-geniculate visual pathway. At present, the pathogenesis of TRDON is secondary apoptosis of P β-type retinal ganglion cells, resulting in the atrophy of optic tract, thinning of the retinal nerve fiber layer and retinal ganglion cell layer thickness and declining of retinal microvascular density, which are consistent with the visual field defect attributed to the primary disease. Of which, the thinning of the retinal ganglion cell layer thickness is considered as the characteristic of TRDON. Now, there is little understanding and related research on TRDON in China. Clinicians should pay attention to the characteristics and severity, occurrence time and location of the above structural changes in these patients through optical coherence tomography, and monitor the activity and progress of the lesions, so as to determine the cut-off point for drug intervention and the drug targets for developing new treatment methods, and bring benefits for patients in partial visual function recovery and disability reduction.
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Objective:To investigate the inhibitory effect of specific inhibitor of necroptosis necrostatin-1 (Nec-1) on necroptosis of retinal ganglion cells (RGCs) in rats with acute ocular hypertension.Methods:Twenty-four adult male Sprague Dawley rats were randomly divided into normal control group, model control group, Nec-1 treatment group and negative control group by random number table method, with 6 rats in each group.High intraocular pressure (IOP)-induced ischemia and reperfusion model was established through anterior chamber irrigation of 0.9% sodium chloride solution in left eyes of the rats, raising the IOP to 110 mmHg (1 mmHg=0.133 kPa) for 60 minutes.Nec-1 (4 mmol/L, 2 μl) or dimethyl sulfoxide (2 μl) was intravitreally injected immediately in Nec-1 treatment group and negative control group following modeling, respectively, according to grouping.No intervention was administered to the normal control group.Paraffin sections of rat retinas of the left eyes in different groups were prepared seven days after modeling.The retinal structure was observed by hematoxylin-eosin staining, and the expression levels of thymocyte antigen-1 (Thy-1) and glial fibrillary acidic protein (GFAP) were detected via immunohistochemical staining.All animal experiments were approved by an Ethics Committee of Tianjin Union Medical Center (No.2017 Quick audit C01).Results:Seven days after modeling, compared with normal control group, the retinal nerve fiber layer was thinner in model control group and negative control group, and the RGCs were arranged loosely, and cells in the inner nuclear layer were reduced and arranged disorderly, and cells in the outer nuclear layer were normal or enlarged.Compared with model control group and negative control group, the nerve fiber layer was thickened and the number of RGCs was significantly increased in Nec-1 treatment group.The number of Thy-1-positive RGCs was decreased in model control group, negative control group and Nec-1 treatment group than normal control group, and there were more Thy-1-positive RGCs in Nec-1 treatment group than model control group and negative control group.The integrated absorbance ( A) value of GFAP protein in normal control group, model control group, negative control group and Nec-1 treatment group was 47.209±15.311, 116.220±18.194, 116.382±19.020, 92.818±10.236, respectively, showing statistically significant differences among them ( F=24.675, P<0.001). The integrated A value of GFAP protein was significantly increased in model control group, negative control group and Nec-1 treatment group than normal control group, and the integrated A value of GFAP protein in Nec-1 treatment group was lower than that in model control group and negative control group, with statistically significant differences (all at P<0.05). Conclusions:Nec-1 can promote RGCs survival by inhibiting the necroptosis of RGCs in rats with acute intraocular hypertension.
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Glaucoma is an irreversible blinding eye disease caused by the structural and functional damage of optic nerve induced by pathological increase of intraocular pressure (IOP), characterized by multiple causes and strong heterogeneity.The control of IOP to reduce the risk of optic damage has been the main therapeutic strategy of glaucoma for many years.However, in clinical experience, some patients show progress of optic nerve damage despite the effectively controlled IOP, the mechanism of non-IOP-dependent secondary damage is still an urgent problem to be solved and a research hotspot in the pathogenesis of glaucoma.With the continuous innovation of molecular biological technology, breakthroughs have been made in the field of basic research.Partial visual recovery can be boosted by alleviating local immune and inflammatory responses.Due to a lack of symbolic clinical application results, it has become an immediate priority to attach importance to the combination of basic clinical research and facilitate the transformation of results.Starting from the theory of glaucoma-immune inflammation, understanding the importance of the immune homeostasis of eyes, paying close attention to the linkage of eyes and brain in physiopathological process and the progression of diseases in the whole visual pathway, and fully understanding and effectively making good use of the opportunities and implications brought by new techniques will have significant effect in formulating clinical diagnosis and treatment plans.
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Objective:To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on the apoptosis of retinal ganglion cells (RGCs) in diabetic retinopathy (DR) model rats and on the regulation of p38 mitogen-activated protein kinase (p38MAPK) pathway.Methods:Forty-five SPF male 8-week old SD rats were selected.The DR rat model was established by intraperitoneal injection of streptozotocin (STZ) combined with a high-sugar and high-fat diet.The fasting blood glucose (FBG) and body weight of the rats were measured every week during the high-sugar and high-fat diet, and fundus angiography was used to observe the circulation and leakage of retinal blood vessels.Forty rats with successful modeling were randomly divided into model group and hUC-MSC injection group according to the random number table method, with 20 rats in each group.Another 20 normal rats fed routinely were served as control group, and intraperitoneally injected with the same amount of citric acid buffer.The hUC-MSC injection group was injected intravitreously with hUC-MSC, and the control group and model group were injected intravitreously with the same amount of phosphate buffer saline (PBS). Fluoro gold (FG) retrograde tracer labeling RGCs was used to observe the number of survived RGCs.Hematoxylin-eosin staining was used to observe the pathological changes of retina.TUNEL method was used to observe the apoptosis of RGCs.Western blot was used to detect B cell lymphoma /leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), p38MAPK and phosphorylated (p-) p38MAPK protein expression in retinal tissues.The use and care of the rats complied with the ARVO statement.The study protocol was approved by an Animal Ethics Committee of Zhengzhou central Hospital Affiliated to Zhengzhou University (NO.2980316).Results:The FBG of control rats was maintained at a normal level, and the body weight gradually increased over time, and was gradually decreased as the course of disease prolonged.The retinal blood vessels ran normally without fluorescein leakage in the control group.In the modeling group, the FBG was maintained at a high level, and the body weight increased slowly and gradually decreased with the prolongation of the disease course since the second week after modeling.The distal retinal vessels were found twisted with large area of capillary fluorescein leakage in the modeling group.The density of RGCs in the control group, model group and hUC-MSC injection group were (2 136.10±215.17), (849.40±167.82), (1 549.20±183.26) cells/mm 2, with significant overall differences ( F=115.218, P<0.01). The density of RGCs in the model group and the hUC-MSC injection group were significantly lower than that of the control group, and the density of RGCs in the hUC-MSC injection group was significantly higher than that of the model group, and the differences were statistically significant (all at P<0.05). The retina in the control group was with clear structure, distinct layers, and a large number of complete RGCs.The number of RGCs in the model group was significantly reduced with nuclear pyknosis, thinned and atrophied RGC layer.The retinal structure was relatively complete, and there were more RGCs in the hUC-MSC injection group than the model group.The apoptosis rates of RGCs in the control group, model group and hUC-MSC injection group were (2.16±1.11)%, (43.47±2.26)%, (20.75±2.18)%, with significant overall difference ( F=445.159, P<0.01). The apoptosis rates of RGCs in the model group and hUC-MSC injection group were significantly higher than that of the control group, and the apoptosis rate of RGCs in the hUC-MSC injection group was lower than that of the model group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of Bax, Bcl-2 and p-p38MAPK proteins in the retina tissues among the three groups ( F=30.982, 12.526, 73.158, all at P<0.01). The relative expression of Bax and p-p38MAPK protein were significantly higher, and the relative expression of Bcl-2 protein was significantly lower in the hUC-MSC injection group and the hUC-MSC injection group than those of the control group, and the differences were statistically significant (all at P<0.05). The relative expression of Bax and p-p38MAPK protein was significantly lower, and the relative expression of Bcl-2 protein was significantly higher in the hUC-MSC injection group than those in the model group, and the differences were statistically significant (all at P<0.05). There was no significant difference in the relative expression of p38MAPK protein among the three groups ( F=1.182, P=0.322). Conclusions:Intravitreal injection of hUC-MSC can inhibit the apoptosis of RGCs in DR model rats and protect the retinal structure of rats, which may play an anti-apoptotic effect by inhibiting the p38MAPK signaling pathway.
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Light therapy, a non-intrusive approach, is now considered as a promising new treatment method for a variety of mood disorders such as depression, bipolar disorder, postpartum depression and so on. However, the neural mechanism of light therapy to regulate emotions is still unclear, and the clinical application of light therapy and its side effects are still controversial. Light therapy regulates mood may be related to the changes of neural circuit mediated by intrinsically photosensitive retinal ganglion cells(ipRGCs), clock gene expression, circadian rhythm and sleep structure. In this paper, the treatment of mood disorders by light has been discussed, and a variety of neural circuits and molecular biological mechanisms of light therapy are introduced, meanwhile, the current situation and side effects of light therapy have been analyzed, in order to provide evidence for the application and promotion of light therapy in the treatment of mood disorders.
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Traumatic optic neuropathy and glaucoma can cause optic nerve degenerative changes leading to a significant decline in vision, which seriously affects the quality of life of patients.In recent years, research on mammalian optic nerve injury models has found that optic nerve injury involves pathophysiological processes such as apoptosis, inflammatory response, and oxidative stress.Researchers have explored the relevant mechanisms and regulatory signaling pathways of optic nerve injury, and have conducted research on the protection of optic nerve injury in the fight against retinal ganglion cell (RGC) apoptosis, new drug therapy, gene therapy, stem cell transplantation and natural extracts.Studies have shown that glucose regulatory protein 75, a member of the heat shock protein 70 family, and melatonin naturally secreted in the human retina may play an important role in the regulation of RGC apoptosis.Human granulocyte colony-stimulating factor (G-CSF) may play a protective role in RGC by directly activating the intrinsic G-CSF receptor and downstream signaling pathway.Targeting gene therapy is expected to become a powerful therapy for repair and regeneration of injured optic nerve.Adipose stem cell transplantation can resist the apoptosis of retinal cells in rat model.In addition, lycium barbarum polysaccharide can delay the secondary degeneration of axons, which may be a promising natural extract to delay the secondary degeneration of optic nerve injury.This article summarized the mechanism, regulation and protection of optic nerve injury.
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Objective:To investigate the effect of pressure on the differentiation of rabbit retinal stem cells (RSCs) co-cultured with retinal ganglion cells (RGCs).Methods:SPF grade New Zealand rabbits on the day 22 of gestation were selected, and embryos were removed to obtain retinal ciliary margin pigment epithelial tissue and culture primary RSCs.Six SPF grade newborn New Zealand rabbits were selected, and retinal neuroepithelial layer tissues were isolated to culture primary RGCs.Rabbit RSCs cultured in vitro were identified by immunofluorescence staining of nestin antibody, bromodeoxyuridine (BrdU) cell proliferation assay kit, RSCs spontaneously differentiated cells immunofluorescence detection and flow cytometry.RGCs were identified through immunofluorescence staining of Brn3b antibody and Thy1.1 antibody.A co-culture system of RGCs and RSCs cultured in the upper and lower layers of a transwelll plate respectively was constructed.The mRNA and protein expression levels of nestin and Thy1.1 in RSCs and differentiated cells under pressures of 0, 20, 40, 60, 80 mmHg (1 mmHg=0.133 kPa) were detected by real-time fluorescence quantitative PCR and Western blot.The feeding and use of laboratory animals were in accordance with the Regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Yunnan University Affiliated Hospital (No.KPRC-IACUC17008). Results:RSCs cultured in vitro were nestin-positive.The percentage of BrdU-positive isolated RSCs was (92.26±3.28)%.Some cells differentiated from RSCs were Brn3b-positive, accounting for (13.00±3.06)%, and some were GS-positive, accounting for (31.60±3.67)%.RGCs cultured in vitro were Brn3b- and Thy1.1-positive.There were statistically significant differences in the relative mRNA and protein expressions of nestin and Thy1.1 between RSCs and differentiated cells under different pressures (mRNA: F=127.600, 137.400; both at P<0.01; protein: F=82.480, 158.700; both at P<0.001). The relative mRNA and protein expressions of nestin were significantly reduced in RSCs, and relative mRNA and protein expressions of Thy1.1 were significantly increased in differentiated cells at 20, 40, 60 and 80 mmHg in comparison with 0 mmHg (all at P<0.05). When the pressure was 40 mmHg, the relative mRNA and protein expressions of nestin were lowest in RSCs, and the relative mRNA and protein expressions of Thy1.1 in differentiated cells were highest. Conclusions:Within a certain range, pressure can promote the differentiation of RSCs co-cultured with RGCs into ganglion-like cells, and excessive pressure can inhibit the differentiation of RSCs.
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Objective:To explore the light response, retinal inflammation and apoptosis of the retinal ganglion cells (RGCs) 1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice. Methods:Twenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group, 12 mice in each group. 1.5 μl of recombinant adeno-associated virus (rAAV)2/2-cytomegalovirus (CMV)- PsCatCh2.0-enhanced green fluorescent protein (EGFP) was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group, and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus. One year after virus injection, the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique; the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining; the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs. Hematoxylineosin staining was performed to measure the inner retinal thickness. Western blotting was used to detect the protein expression of nuclear factor (NF)-κB p65 in retina; real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and Bax mRNA. Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice. Results:One year after the intravitreal injection of recombinant virus, PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent. The virus PsCatCh2.0-EGFP was mainly transfected into RGCs, and partly transfected into amacrine cells, almost no transfection was seen in bipolar and horizontal cells. There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group ( F=14.35, 0.05; P>0.05), while the rd1 experimental group NF-κB p65 protein expression, TNF-α and IL-6 mRNA quantification were significantly lower than those of rd1 control group ( F=4.61, 5.91, 5.78; P<0.05). The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group, and the Bax mRNA expression was lower than that in the rd1 control group, and the difference was statistically significant ( F=7.52, P<0.01). Conclusion:One year after intravitreal injection of recombinant virus, the PsCatCh2.0 expressing RGCs can still generate photocurrent. Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs, nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.
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Objective:To investigate the effect of erigeron breviscapus (EBHM) on ocular hypertension and the protective effect of retinal ganglion cells (RGCs) in rats by regulating mitogen activated protein kinase (MAPK) signaling pathway.Methods:Sixty male Sprague-Dawley rats were divided into control group, model group, low-dose EBHM group (group A), medium-dose EBHM group (group B), and high-dose EBHM group (group C) by random number table method. There were 12 rats in the group, the left eye was used as the experimental eye. The rats of model group, group A, group B, and group C were infused with normal saline through the anterior chamber to construct an acute ocular hypertension model; the control group was given general anesthesia only. Then, 2-30 days after modeling, rats in the control group and model group were given 3 ml of normal saline once a day; rats in group A, group B, and group C were given 0.30, 0.45, and 0.60 g/100 g EBHM by intragastric administration, respectively, 1 time/d. The rat intraocular pressure was measured before modeling and 1, 14, and 30 days after modeling, and the proportion of high intraocular pressure model was measured. Thirty days after modeling, hematoxylin-eosin (HE) staining was used to observe the pathological changes of retinal tissue; immunofluorescence staining was used to detect the changes in the number of RGCs; real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect p38 in the retinas of rats in each group. The relative expression of MAPK and Caspase-3 mRNA; western blot was used to detect p38MAPK and phosphorylation in the retina of rats in each group relative expression of phosphorylate-p38MAPK (p-p38MAPK) and Caspase-3 protein. One-way analysis of variance was used for multi-sample comparison, and SNK-q test was used for comparison between two samples. Results:One day after modeling, none of the rats in the control group developed acute ocular hypertension, and the other groups were successfully modeled. Compared with the model group, the rates of acute ocular hypertension at 14 days after modeling in groups B and C were lower ( χ2=98.701, P<0.05), and the rates of acute ocular hypertension at 30 days after modeling in groups A, B, and C were 0. There was no statistically significant difference in the rates of acute ocular hypertension between 14 and 30 days after modeling in the A, B, and C groups ( P>0.05). The results of HE staining showed that the structure of the retina in the control group was complete, and the layers were clearly visible; the RGCs count was not abnormal, and the morphology was plump and round. The retina of rats in the model group became thinner; the number of RGCs was greatly reduced, the morphology was vacuolated, and the arrangement was sparse. The retina of rats in groups A, B, and C became thicker, and the number of RGCs increased, and the retina structure in group C was better restored. The results of immunofluorescence staining showed that the RGCs counts of rats in groups A, B, and C were higher than those in the model group, and the difference was statistically significant ( F=297.514, P<0.05); pairwise comparison between groups, group A was lower than that of group B and C Group ( q=2.842, 5.263), group B was lower than group C ( q=2.457), the difference was statistically significant ( P<0.05). The results of RT-qPCR and Western blot showed that compared with the model group, the relative expression of Caspase-3 mRNA ( F=267.912) and protein ( F=692.279) and the relative expression of p-p38MAPK protein in the retina of rats in groups A, B and C. The expression level ( F=150.061) all decreased, and the difference was statistically significant ( P<0.05); pairwise comparisons between groups showed that Caspase-3 mRNA ( q=6.977, 15.642) and protein ( q=6.997, 15.642) relative expression levels and p-p38MAPK protein ( q=12.443, 24.358) relative expression levels are lower than groups A and B, group B was lower than group A ( q=11.678, 12.471, 10.204), the difference was statistical academic significance ( P<0.05). Conclusions:EBHM can significantly reduce intraocular pressure in rats with acute ocular hypertension, increase RGCs counts, and reduce retinal damage. Its regulatory mechanism may be related to the MAPK pathway.
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Objective:To investigate the survival rate change of retinal ganglion cells（RGCs）in a mouse of optic nerve crush（ONC）.Methods:Ninety-seven male C57BL/6J mice（6 to 8 weeks）were selected and divided into normal group（ n=5）, sham-operation group（ n=5）and ONC group（ n=5）according to the random number table. In normal group, both eyes of the mice did not receive any intervention. In sham-operation group, the right eye of the mice received sham operation, while the left eye reveived no intervention. In ONC group, the left eye received ONC, and the right eye received sham operation. In normal group, the density of RGCs in both eyes was quantified and compared. In sham-operatioin group, the density of RGCs in the sham operation eye was calculated and then compared to the average density of RGCs in normal group. In ONC group, the survival rate of RGCs was set as the ratio between the left eye（ONC eye）and the right eye（sham-operation eye）. The survival rate of RGCs in ONC group was compared after crush injury for 5, 10, 20, 30 seconds）（the sacrifice time was set at day 7）, and was compared after sampling on days 3, 4, 5, 7, 14, 30, 60, 90, 180（the duration of crush injury was set as 20 seconds）. Results:In normal group, the density of RGCs in the right eye was（5, 167.3±55.6）cell/mm 2, with no statistical difference from that in the left eye［（5, 199.6±44.8）cell/mm 2］（ P>0.05）. The density of RGCs in normal group and sham-operation group was（5, 183.5±33.4）cell/mm 2 and（5, 151.5±87.6）cell/mm 2, showing no statistical difference（ P>0.05）. The survival rate of RGCs in ONC group after crush injury for 5, 10, 20, 30 seconds was（37.6±1.1）%,（34.0±0.9）%,（33.6±1.6）% and（30.3±0.6）%（ P<0.01）. In comparison, there was statistical difference in the survival rate of RGCs between crush injury for 5 seconds and for 30 seconds（ P<0.01）, but not among other duration of crush injury（ P>0.05）. The survival rate of RGCs in ONC group after sampling on days 3, 4, 5, 7, 14, 30, 60, 90, 180 was（85.4±2.0）%,（67.6±3.1）%,（43.0±1.0）%,（33.6±1.6）%,（22.7±2.0）%,（12.8±0.6）%,（10.4±0.8）%,（8.6±0.5）% and（6.7±0.2）%（ P<0.01）, showing the most obvious drop from day 3 to day 5. Additionally, the curve became flattened after 30 days. Conclusions:In a mouse model of ONC, varying durations of crushing will lead to different damage to RGCs in a progressive mode, indicating that following the primary injury（ONC）, the RGCs suffer secondary injury as well. Therefore, effectively controlling the secondary injury may be the key point of treating optic nerve injuries.