Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats.

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue (EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley (SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12wk and the diabetic groups for 4, 8, and 12wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation. RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was (0.54±0.23)%, (0.65±0.11)%, and (0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%, (0.03±0.05)%, and (0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were (0.53±0.22)%, (0.69±0.16)%, and (0.52±0.11)%. The leakage areas of deep blood vessels were (0.54±0.50)%, (1.42±0.16)%, and (1.80±0.07)% at 4, 8, and 12wk, respectively. There was a statistically difference of the leakage area between the 8th week and the 4th week of diabetes group (P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4wk and 8wk (P<0.001). CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Mingmu Xiaomeng Tablets Restore Autophagy and Alleviate Diabetic Retinopathy by Inhibiting PI3K/Akt/mTOR Signaling.

Objective: To investigate the effect of Mingmu Xiaomeng tablets (MMXM) on the expression of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)-related proteins in a diabetic rat model. Methods: Thirty-two male Sprague Dawley rats were randomly divided into four groups: normal control (NC), diabetic model (DM) control, MMXM, and calcium dobesilate (CD) Rats injected with streptozotocin (STZ) were used as an experimental diabetes model. After 14 weeks, autophagy and PI3K/Akt/mTOR signaling pathway proteins were detected by western blot. Glial fibrillary acidic protein (GFAP) expression in Müller cells was examined by immunohistochemistry. Retinal function was evaluated with electroretinography, and retinal ultrastructure was observed by transmission electron microscopy. Serum cytokine levels were detected with protein chip technology. Results: MMXM restored autophagy by decreasing the protein expression of LC3-II and p62 and reducing the phosphorylation of PI3K, Akt, and mTOR, thus promoting autophagy. MMXM decreased GFAP expression in retinal Müller cells; restored electrophysiology indexes and retinal ultrastructures; and reduced serum levels of interleukin (IL)-1ß, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor. Conclusion: MMXM may protect the diabetic retina by inhibiting PI3K/Akt/mTOR signaling and enhancing autophagy.

2,3,5,6-Tetramethylpyrazine protects retinal photoreceptors against endoplasmic reticulum stress by modulating ATF4-mediated inhibition of PRP aggregation.

Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: • Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. • New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. • Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.

Activation of the TRAAK two-pore domain potassium channels in rd1 mice protects photoreceptor cells from apoptosis.

AIM: To investigate the expression of TWIK-related arachidonic acid-stimulated K+ channel (TRAAK) in retinal degeneration mice (rd1) and further evaluate how TRAAK affect photoreceptor cell apoptosis. METHODS: The rd1 mice were distributed into blank (no treatment), control (1.4% DMSO, intraperitoneal injection) and riluzole groups (4 mg/kg·d, intraperitoneal injection) from postnatal 7d to 10, 14 and 18d; C57 group (no treatment), as age-matched wild-type control. The thickness of the outer nuclear layer (ONL) of retina was detected by paraffin section hematoxylin and eosin staining. The expression of TRAAK and the apoptosis of the ONL cells were detected by immunostaining, Western blotting, and real-time polymerase chain reaction. RESULTS: The channel agonist riluzole activated TRAAK and delayed the apoptosis of photoreceptor cells in ONL layer of rd1 mice. Both at mRNA and protein levels, after riluzole treatment, TRAAK expression was significantly upregulated, when compared with the control and blank group. Then we detected a series of apoptosis related mRNA and protein. The anti-apoptotic factor Bcl-2 downregulated and the pro-apoptotic factors Bax and cleaved-caspase-3 upregulated significantly. CONCLUSION: Riluzole elevates the expression of TRAAK and inhibits the development of apoptosis. Activation of TRAAK may have some potential effects to put off photoreceptor apoptosis.

Elevated expression of TREK-TRAAK K2P channels in the retina of adult rd1 mice.

AIM: To examine the expression of Twik-related K+ channel 1 (TREK-1), Twik-related K+ channel 2 (TREK-2), and Twik-related arachidonic acid-stimulated K+ channel (TRAAK) in the retina of adult rd1 mice and to detect the protective roles of TREK-TRAAK two-pore-domain K+ (K2P) channels against retinal degeneration. METHODS: Twenty-eight-day-old C57BL/6J mice and 28-day-old rd1 mice were used in this study. Retinal protein, retinal RNA, and embedded eyeballs were prepared from these two groups of mice. Real-time quantitative polymerase chain reaction and Western blot analyses were used to assess the gene transcription and protein levels, respectively. Retinal structures were observed using hematoxylin and eosin (H&E) staining. Immunohistochemistry was utilized to observe the retinal localization of TREK-TRAAK channels. Current changes in retinal ganglion cells (RGCs) after activation of TREK-TRAAK channels were examined using a patch-clamp technique. RESULTS: Compared with C57BL/6J mice, rd1 mice exhibited significantly higher retinal mRNA and protein expression levels of TREK-1, TREK-2, and TRAAK channels. In both groups, immunohistochemistry showed expression of TREK-TRAAK channels in retinal layers. After addition of the TREK-TRAAK channel agonist arachidonic acid (AA), whole-cell voltage step evoked currents were significantly higher in RGCs from rd1 mice than in RGCs from control C57BL/6J mice, suggesting that TREK-TRAAK channels were opened in RGCs from rd1 mice. CONCLUSION: TREK-TRAAK K2P channels' expression is increased in adult rd1 mice. AA induced the opening of TREK-TRAAK K2P channels in adult rd1 mice and may thus counterbalance depolarization of RGCs and protect the retina from excitotoxicity. TREK-TRAAK channels may play a protective role against retinal degeneration.

INTRAVITREAL GAS INJECTION WITH LASER PHOTOCOAGULATION FOR HIGHLY MYOPIC FOVEOSCHISIS: Technique and Outcome.

PURPOSE: To evaluate the effects of gas tamponade combined with laser photocoagulation without vitrectomy in patients with highly myopic foveoschisis. METHODS: This retrospective noncomparative case series included 30 eyes of 23 patients with highly myopic foveoschisis who were treated by intravitreal injection of 0.5 to 0.7 mL C3F8 combined with laser photocoagulation 1 week later, and remained in the face-down position for 3 weeks. The patients were followed up for at least 6 months after the treatment. The refractive status, best-corrected visual acuity, and anatomical parameters of retina observed with the optical coherence tomography before and after the treatment were recorded. RESULTS: The mean age of the 23 patients (6 men and 17 women) was 50.4 ± 15.1 years. The average follow-up duration was 20.8 ± 20.6 months. At the final follow-up, 23 eyes (76.7%) completely (17 eyes) or partially (6 eyes) resolved. Seventeen eyes had complete data of optical coherence tomography parameters, the mean central foveal thickness decreased significantly from 505.24 ± 466.22 µm to 186.41 ± 95.36 µm (P = 0.01), and the mean maximal macular thickness from 687.88 ± 397.00 µm to 313.65 ± 83.07 µm (P = 0.001). The mean final logarithm of the minimum angle of resolution best-corrected visual acuity (Snellen equivalent) of the 30 eyes ranged from 1.6 (20/800) to 0.2 (20/32), showing a slight improvement from 0.91 ± 0.44 (20/163) preoperatively to 0.90 ± 0.39 (20/160) postoperatively (P = 0.87). CONCLUSION: C3F8 tamponade combined with laser photocoagulation could be an alternative treatment for highly myopic foveoschisis.

TREK­TRAAK two­pore domain potassium channels protect human retinal pigment epithelium cells from oxidative stress.

The aim of the current study was to explore the potential of TREK­TRAAK two­pore domain potassium (K2P) channels in protecting human retinal pigment epithelium (hRPE) cells against oxidative stress. hRPE cells were obtained from donors, and then cell identification and detection of the expression levels of TREK­TRAAK K2P channels in hRPE cells were conducted. Subsequently, tert­butyl hydroperoxide (t­BH) was used to induce oxidative stress in hRPE cells. Docosahexaenoic acid (DHA) was used to stimulate and fluoxetine was used to inhibit the TREK­TRAAK K2P channels. The survival rates of hRPE cells under oxidative stress were examined using flow cytometry. Apoptosis­associated factors, including Bax, Bcl­2, cleaved­caspase­3, αB­crystallin and their mRNAs, were examined using immunofluorescence, western blot and reverse transcription­polymerase chain reaction analyses. Variations in the cytoarchitecture were observed by immunofluorescence and electron microscopy. The cells examined in the present study were identified as hRPE cells. All members in the TREK­TRAAK K2P channel family (including TREK­1, TREK­2 and TRAAK) were found to be expressed in hRPE cells. Stimulation of TREK­TRAAK K2P channels increased the survival rates of hRPE cells under oxidative stress and the levels of intracellular protective factors, such as Bcl­2 and αB­crystallin. By contrast, inhibition of these channels decreased the cell survival rates and increased apoptosis enhancing factors, such as Bax and cleaved­caspase­3. Further examination of the cytoarchitecture revealed that TREK­TRAAK K2P channels protected the integrity of the hRPE cell structure against oxidative stress. In conclusion, the present study suggested that the activated TREK­TRAAK K2P channels serve a role in protecting hRPE cells against the oxidative stress induced by t­BH, which indicated that these K2P channels are potential novel targets in retinal protection and provided a new direction for research and therapy in retinal degeneration diseases.