The glutamate excitotoxicity has been suggested as a factor involved in the loss of retinal neuronal cells, including retinal ganglion cell (RGC), in various retinal degenerative diseases including ...ischemia-reperfusion injury, diabetic retinopathy, and glaucoma. Excitotoxic RGC death is caused not only by direct damage to RGCs but also by indirect damage due to the inflammation of retinal glial cells. Sphingosine 1-phosphate (S1P) and ceramides are bioactive sphingolipids which have been shown to possess important physiological roles in cellular survival and apoptosis, and the balance between S1P and ceramide, sphingolipid rheostat, has been suggested to be important for determining cellular fate. Therefore, we conducted the present study to clarify the neuroprotective role of sphingolipid rheostat in excitotoxic RGC death in vivo and in vitro. Acute RGC death was induced by intravitreal N-methyl-d-aspartate (NMDA) injection in the mouse. The mRNA expression of sphingosine kinase (SphK1/SphK2) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of SphK1/2, S1P, S1P-receptor (S1PR), glial fibrillary acidic protein (GFAP), Iba1, and CD31 were examined by immunostaining. Retinal sphingolipids and ceramides were quantified by liquid chromatography with tandem mass spectrometry. The neuroprotective effect of the sphingosine kinase inhibitor (SKI) on RGC death was assessed by RGC count and Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Further, the in vitro effect of SKI was investigated using rat primary cultured RGCs and glial cells. In addition, MG5 cells and A1 cells, which were mouse microglia and astrocyte cell-line, were also used. The expression of cleaved-caspase-3, GFAP, and Iba1 in RGCs, primary glial cells, MG5 cells, and A1 cells was assessed by immunostaining. NMDA injection resulted in mRNA upregulation of SphK1; however, SphK2 was reduced in the mouse retina. SphKs, S1P, S1PR1, S1PR2, and GFAP expression increased in the early-stage NMDA group, whereas S1P and GFAP were higher in the late-stage NMDA + SKI group. In the NMDA group, S1P expression was lower whereas sphingosine, C20, C22, and C24 ceramides showed higher levels. The proportion of very-long-chain ceramide was elevated in the NMDA group but reduced in the NMDA + SKI group. SKI treatment significantly increased RGC survival in retinal wholemount analysis and decreased apoptosis in the ganglion cell layer and inner nuclear layer. In vitro, SKI suppressed excitotoxic RGC death, cleaved-caspase-3 expression, and activated glial cells. The findings in the present study provide the first evidence demonstrating the involvement of sphingolipid rheostat in the neuroprotection against excitotoxic RGC death. Therefore, regulation of sphingolipid rheostat might serve as a potential therapy for retinal degenerative disease.
•S1P -ceramide balance, sphingolipid rheostat, play a pivotal role in retinal ganglion cells (RGCs).•SKI suppressed the increase of very-long chain ceramide in retinal glutamate excitotoxicity.•The neuroprotective effect of SKI was exerted directly on RGCs and indirectly through glial cells.
Abstract
This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm’s canal endothelial (SCE) cells, and ...porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.
To determine the course of occult macular dystrophy (OMD, Miyake's disease) and to propose stages of OMD based on the optical coherence tomographic (OCT) findings.
Sixty-one patients from 33 families ...with OMD who carried one of the proven variants of the RP1L1 gene were studied at seven centers in Japan. Ophthalmological examinations including the best-corrected visual acuity (BVCA) and OCT were performed.
The median age at the last visit was 50 years with a range of 10 to 88 years, and the median age at the symptom onset was 30 years with a range of 3 to 60 years. There were significant negative correlations between the duration of OMD and BCVA, the central retinal thickness (CRT) and the thickness between external limiting membrane and retinal pigment epithelium (ERT). The BCVA gradually decreased for 10 years after symptom onset and was stable thereafter. Kaplan-Meier survival curves of the BCVA and retinal thickness showed that all of the patients had retained a vision of 1.0 logMAR, and over 80% of the patients had retained 50% thickness of the normal CRT and ERT for at least 60 years after symptom onset. The stages of OMD based on the visual symptoms and OCT findings are proposed.
The photoreceptors do not become completely atrophic even at the late stage, which may account for the good retinal pigment epithelium (RPE) structure and normal-appearing fundus. The proposed stages facilitate the investigation of the pathogenicity of OMD and provide information to determine the effectiveness of therapeutic procedures.
Purpose
To examine the effect of switching from a prostanoid FP receptor agonists to EP2 receptor agonist (omidenepag isopropyl) on the deepening of the upper eyelid sulcus (DUES) and intraocular ...pressure (IOP) in Japanese glaucoma patients over 3 months post treatment.
Study design
Prospective observational study.
Methods
Patients with glaucoma who received FP receptor agonists treatment and had complained of DUES-related reduction in quality of life were included. Their FP receptor agonists was switched to omidenepag isopropyl without a drug holiday. At baseline and 1 and 3 months post-switch, photographs were taken and the changes in DUES were assessed by three independent observers. IOP and adverse events were also assessed.
Results
The study included 23 eyes of 23 patients (6 men, 17 women; average age, 60.6 years). After switching, DUES improved in 12 eyes at 1 month and in 16 eyes at 3 months; eyes in the remaining patients showed no worsening of the condition. The mean IOP before switching was 15.3 ± 3.3 mmHg (95% confidence interval 13.9–16.7 mmHg). Following the switch, the mean IOP values were 15.6 ± 3.3 mmHg (14.1–17.0 mmHg) at 1 month and 15.5 ± 3.3 mmHg (14.1–16.9 mmHg) at 3 months (P = 1.0 at 1 month, P = 1.0 at 3 months; both adjusted by Bonferroni correction). No adverse effects were observed.
Conclusion
Omidenepag isopropyl improved DUES while maintaining IOP in over 70% of Japanese patients with glaucoma who exhibited DUES caused by FP receptor agonists; the improvement was observed within 3 months after switching from FP receptor agonists.
We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. ...We analyzed 514 embryos monitored with TLM 6-9 h after insemination and 144 embryos monitored using microscopic assessment 18-21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within < 20 h following the initiation of insemination. The incidence of 0PN-derived embryo reduced more significantly through TLM than through fixed-point observation. The microscopic assessment time was more significantly delayed in the 0PN-derived embryo than that in the 2PN-derived embryo. The embryo with early PN fading formed good-quality blastocysts, and their pregnancy outcomes were similar to those of other embryos. Most 0PN-derived embryos in the fixed-point assessment might have resulted from missed observation of PN appearance in the early-cleaved embryos. TLM or strict laboratory schedule management may reduce 0PN-derived embryos by reducing missed PN observations.
ObjectivesHuman chorionic gonadotropin (hCG) levels are essential for the management of trophoblastic diseases. This study aimed to compare the sensitivities and relationships of two hCG measurement ...methods (total hCG and the free β-subunit of hCG) in managing gestational trophoblastic disease (GTD).Design and MethodsWe analyzed data from patients treated for GTD at Chiba University Hospital between 2008 and 2019. We focused on cases where both total hCG (mIU/mL) and the free β-subunit of hCG (ng/mL) were measured on the same day.ResultsOut of 80 patients (mean age 38.9 ± 11.7 years) and 158 measurements, 26 had values below the sensitivity threshold for both tests. Fifty-nine measurements were positive for total hCG but below the sensitivity threshold for the free β-subunit of hCG, whereas only two showed the opposite. Seventy-one measurements were positive for both total hCG and the free β-subunit of hCG. There was a significant correlation between total hCG and the free β-subunit of hCG with both positive values, (r = 0.94, p < 0.001; Spearman's correlation test). Of the 85 measurements with undetectable free β-subunit levels, 26 also had undetectable total hCG levels. However, total hCG was detectable in 59 patients from these cases, with a median value (interquartile range) of 2.9 (1.75-4.9) mIU/mL.ConclusionsIn the management of GTD, the use of the free β-subunit system alone cannot be recommended.
We analyzed the effects of enzyme replacement therapy (ERT) on the visual acuity and visual fields of a patient with mucopolysaccharidosis type II, Hunter syndrome, with degeneration of the retina ...and abnormalities of the optic nerve. After the ERT, there was an improvement of the visual acuity and visual fields and an improvement of the activities of daily living. Despite the late onset of Hunter syndrome in this patient, ERT was still able to improve the visual function. We conclude that ERT should be considered regardless of the age of the manifestations of the signs and symptoms of Hunter syndrome.
This study examines the potential role of transforming growth factor-beta 3 (TGF-β3) on the fibrotic response of cultured human trabecular meshwork (HTM) cells. The relationships and trans-signaling ...interactions between TGF-β3 and autotaxin (ATX) in HTM cells were also examined. The levels of TGF-β and ATX in the aqueous humor (AH) of patients were measured by an immunoenzymetric assay. The TGF-β3-induced expression of the fibrogenic markers, fibronectin, collagen type I alpha 1 chain, and alpha-smooth muscle actin, and ATX were examined by quantitative real-time PCR, Western blotting, and immunocytochemistry, and the trans-signaling regulatory effect of TGF-β3 on ATX expression was also evaluated. In HTM cells, the significant upregulation of ATX was induced by TGF-β3 at a concentration of 0.1 ng/mL, corresponding to the physiological concentration in the AH of patients with exfoliative glaucoma (XFG). However, higher concentrations of TGF-β3 significantly suppressed ATX expression. TGF-β3 regulated ATX transcription and signaling in HTM cells, inducing the upregulation of fibrogenic proteins in a dose-dependent manner. Trans-signaling of TGF-β3 regulated ATX transcription, protein expression, and signaling, and was thereby suggested to induce fibrosis of the trabecular meshwork. Modulation of trans-signaling between TGF-β3 and ATX may be key to elucidate the pathology of XFG, and for the development of novel treatment modalities.
The retinitis pigmentosa 2 (RP2) gene is one of the causative genes for X‐linked inherited retinal disorder. We characterized the clinical/genetic features of four patients with RP2‐associated ...retinal disorder (RP2‐RD) from four Japanese families in a nationwide cohort. A systematic review of RP2‐RD in the Japanese population was also performed. All four patients were clinically diagnosed with retinitis pigmentosa (RP). The mean age at examination was 36.5 (10–47) years, and the mean visual acuity in the right/left eye was 1.40 (0.52–2.0)/1.10 (0.52–1.7) in the logarithm of the minimum angle of resolution unit, respectively. Three patients showed extensive retinal atrophy with macular involvement, and one had central retinal atrophy. Four RP2 variants were identified, including two novel missense (p.Ser6Phe, p.Leu189Pro) and two previously reported truncating variants (p.Arg120Ter, p.Glu269CysfsTer3). The phenotypes of two patients with truncating variants were more severe than the phenotypes of two patients with missense variants. A systematic review revealed additional 11 variants, including three missense and eight deleterious (null) variants, and a statistically significant association between phenotype severity and genotype severity was revealed. The clinical and genetic spectrum of RP2‐RD was illustrated in the Japanese population, identifying the characteristic features of a severe form of RP with early macular involvement.
Full-field electroretinograms (ERGs) are used to evaluate retinal function in patients with various types of hereditary and acquired retinal diseases. However, ERG recordings require relatively ...invasive procedures, including pupillary dilation and the use of contact lens electrodes. Thus, it would be helpful to have a simpler and noninvasive screening method. The purpose of this study was to determine whether a new, handheld, portable ERG device, RETeval™, can be used to screen patients for cone dysfunction.
Thirty-five eyes of 35 patients who had reduced cone responses ascertained by a conventional ERG system using contact lens electrodes were studied. The causative diseases included achromatopsia, cone dystrophy, cone-rod dystrophy, retinitis pigmentosa, choroidal dystrophy, autoimmune retinopathy, and Stargardt disease. The flicker ERGs were recorded with the RETeval™ under undilated conditions with skin electrodes (stimulus strength, 3.0 cd·s/m(2); frequency, 28.3 Hz), and the responses were compared to that of 50 healthy eyes. The amplitudes and implicit times of the fundamental component of the flicker ERGs were analyzed in three age groups: Group A, ≤20 years; Group B, 21-40 years; and Group C, ≥41 years.
In all of the age groups, the amplitudes of the ERGs were significantly smaller and the implicit times significantly longer in patients with cone dysfunction than in the control eyes. All but one of the patients had flicker amplitudes lower than the mean -2.0 standard deviation of control eyes.
The RETeval™ has a potential of being used to screen for cone dysfunction. The entire examination takes <5 minutes and does not require pupil dilatation or a contact lens electrode. Although the flicker responses do not provide information on the scotopic functions, the RETeval™ device can be used to determine which patients require additional full-field ERG testing with dilated pupils under both scotopic and photopic conditions.
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