Xi Ying

α-Crystallin Downregulates the Expression of TNF-α and iNOS by Activated Rat Retinal Microglia in vitro and in vivo

Purpose: Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of α-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia. Methods: Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without α-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method. Results: Activated microglia significantly upregulated TNF-α and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-α protein. Treatment with α-crystallin in vitro and in vivo downregulated their expression. Conclusion: The protective effect of α-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-α and iNOS.

α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush

AIMS Activation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush. MAIN METHODS RGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05g/kg, 0.5g/kg, and 5 g/kg) at 2, 4, 6, 8, 10, and 12 days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4 weeks after injury using fluorescence and confocal microscopy. KEY FINDINGS The number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury. SIGNIFICANCE Our results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury.

Promotion of axon regeneration and inhibition of astrocyte activation by alpha A-crystallin on crushed optic nerve.

AIM To explore the effects of αA-crystallin in astrocyte gliosis after optic nerve crush (ONC) and the mechanism of α-crystallin in neuroprotection and axon regeneration. METHODS ONC was established on the Sprague-Dawley rat model and αA-crystallin (10(-4) g/L, 4 µL) was intravitreously injected into the rat model. Flash-visual evoked potential (F-VEP) was examined 14d after ONC, and the glial fibrillary acidic protein (GFAP) levels in the retina and crush site were analyzed 1, 3, 5, 7 and 14d after ONC by immunohistochemistry (IHC) and Western blot respectively. The levels of beta Tubulin (TUJ1), growth-associated membrane phosphoprotein-43 (GAP-43), chondroitin sulfate proteoglycans (CSPGs) and neurocan were also determined by IHC 14d after ONC. RESULTS GFAP level in the retina and the optic nerve significantly increased 1d after ONC, and reached the peak level 7d post-ONC. Injection of αA-crystallin significantly decreased GFAP level in both the retina and the crush site 3d after ONC, and induced astrocytes architecture remodeling at the crush site. Quantification of retinal ganglion cell (RGC) axons indicated αA-crystallin markedly promoted axon regeneration in ONC rats and enhanced the regenerated axons penetrated into the glial scar. CSPGs and neurocan expression also decreased 14d after αA-crystallin injection. The amplitude (N1-P1) and latency (P1) of F-VEP were also restored. CONCLUSION Our results suggest α-crystallin promotes the axon regeneration of RGCs and suppresses the activation of astrocytes.

Morphometric Measurements of Fetal and Neonatal Eyes Using MRI and Ultrasound

Using MRI, the ratios of the anterior posterior (AP) lengths of the eyes versus the AP lengths of the corneae to the external occipital protuberances were found to decrease from 28 weeks of gestation onwards, while the ratios of the transverse diameters of the eyes versus the transverse diameters of the zygomatic bone to the nasal cavity increased till term. No further change in the ratios of the AP or transverse diameters was observed even till three years of age.

Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons

AIM To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. MAIN METHODS Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. KEY FINDINGS Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (P<0.05). Moreover, endogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (P<0. 01) and was associated with a decrease in caspase-3 expression (P<0. 05). SIGNIFICANCE The present study demonstrates that the expression of endogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons.

Comparison of Retinal Degeneration in the Normal Goldfish and the Megalophthalmic Black Moor Goldfish after Optic Nerve Transection and Lens Extraction

A comparison in retinal degeneration was studied in the normal goldfish and the megalophthalmic goldfish after optic nerve transection or lens extraction by the TUNEL (Terminal transferase-mediated dUTP nick end labeling) technique. A significant number of TUNEL positive cells appeared in both cases 7 days after injury, with a more prominent result in the megalophthalmic eye. Lens extraction, had less apoptotic cell death in the experimental retinas.

Use of Fluorescence Fundus Angiography to Assess Variations in Retinal Circulation in Myopic Patients of Different Ages

To investigate the variation in retinal circulation in patients with different degrees of myopia and different age by fluorescence fundus angiography (FFA). Thirty myopic patients (60 eyes), 20–54 years of age, with various degrees of myopia were evaluated. All patients underwent fundus photography and FFA using a standard technique. Patients with other ophthalmologic diseases possibly affecting the retrobulbar circulation and those with a history of laser treatment or intraocular surgery were excluded. Vascular filling patterns at arterial phase, arterial-venous phase and venous phase were reviewed. The average filling times at each phase were significantly delayed in severe myopia, but there was no difference between eyes with mild and medium myopia. There was no statistically significant correlation between mean filling time and age. The findings indicated a significant correlation between the intraocular blood circulation and the degree of myopia.